Whole-genome sequencing of two captive black soldier fly populations: Implications for commercial production.

Black soldier fly (BSF; Hermetia illucens) is a promising insect species for food and feed production as its larvae can convert different organic waste to high-value protein. Selective breeding is one way to optimize production, but the potential of breeding is only starting to be explored and not yet utilized for BSF. To assist in monitoring a captive population and implementing a breeding program, genomics tools are imperative. We conducted whole genome sequencing of two captive populations separated by geographical distance - Denmark (DK) and Texas, USA (TX). Various population genetics analyses revealed a moderate genetic differentiation between two populations. Moreover, we observed higher inbreeding in the DK population, and the detection of a subpopulation within DK population aligned well with the recent foundation of the DK population from two captive populations. Additionally, we generated gene ontology annotation and variants annotation for wider potential applications. Our findings establish a robust marker set for research in population genetics, facilitating the monitoring of inbreeding and laying the groundwork for practical breeding programs for BSF.

Diurnal variation in genetic parameters for locomotor activity in Drosophila melanogaster assessed under natural thermal conditions.

In nature, organisms are exposed to variable and occasionally stressful environmental conditions. Responses to diurnal and seasonal fluctuations, such as temperature and food accessibility, involve adaptive behavioural and physiological changes. While much work has been done on understanding the genetic architecture and evolutionary potential of stress tolerance traits under constant thermal conditions, there has been less focus on the quantitative genetic background in variable environments. In this study, we use the Drosophila Genetic Reference Panel (DGRP) to investigate the locomotor activity, a key behavioural trait, under variable natural thermal conditions during the summer in a temperate environment. Male flies from 100 DGRP lines were exposed to natural thermal and light conditions in Drosophila activity monitors across three experimental days. We found that activity was highly temperature and time dependent and varied between lines both within and between days. Furthermore, we observed variation in genetic and environmental variance components, with low to moderate estimates of the heritability for locomotor activity, consistently peaking in the afternoons. Moreover, we showed that the estimated genetic correlations of locomotor activity between two time points decreased, as the absolute differences in ambient temperature increased. In conclusion, we find that the genetic background for locomotor activity is environment specific, and we conclude that more variable and unpredictable future temperatures will likely have a strong impact on the evolutionary trajectories of behavioural traits in ectotherms.

Strong acclimation effect of temperature and humidity on heat tolerance of the Arctic collembolan Megaphorura arctica.

The Arctic is a highly variable environment in which extreme daily and seasonal temperature fluctuations can occur. With climate change, an increase in the occurrence of extreme high temperatures and drought events is expected. While the effects of cold and dehydration stress on polar arthropods are well studied in combination, little is known about how these species respond to the combined effects of heat and dehydration stress. In this paper, we investigated how the heat tolerance of the Arctic collembola Megaphorura arctica is affected by combinations of different temperature and humidity acclimation regimes under controlled laboratory conditions. The effect of acclimation temperature was complex and highly dependent on both acclimation time and temperature, and was found to have a positive, negative or no effect depending on experimental conditions. Further, we found marked effects of the interaction between temperature and humidity on heat tolerance, with lower humidity severely decreasing heat tolerance when the acclimation temperature was increased. This effect was more pronounced with increasing acclimation time. Lastly, the effect of acclimation on heat tolerance under a fluctuating temperature regime was dependent on acclimation temperature and time, as well as humidity levels. Together, these results show that thermal acclimation alone has moderate or no effect on heat tolerance, but that drought events, likely to be more frequent in the future, in combination with high temperature stress can have large negative impacts on heat tolerance of some Arctic arthropods.

Estimation of genetic parameters for the implementation of selective breeding in commercial insect production.

BACKGROUND: There is a burgeoning interest in using insects as a sustainable source of food and feed, particularly by capitalising on various waste materials and by-products that are typically considered of low value. Enhancing the commercial production of insects can be achieved through two main approaches: optimising environmental conditions and implementing selective breeding strategies. In order to successfully target desirable traits through selective breeding, having a thorough understanding of the genetic parameters pertaining to those traits is essential. In this study, a full-sib half-sib mating design was used to estimate variance components and heritabilities for larval size and survival at day seven of development, development time and survival from egg to adult, and to estimate correlations between these traits, within an outbred population of house flies (Musca domestica), using high-throughput phenotyping for data collection. RESULTS: The results revealed low to intermediate heritabilities and positive genetic correlations between all traits except development time and survival to day seven of development and from egg to adulthood. Surprisingly, larval size at day seven exhibited a comparatively low heritability (0.10) in contrast to development time (0.25), a trait that is believed to have a stronger association with overall fitness. A decline in family numbers resulting from low mating success and high overall mortality reduced the amount of available data which resulted in large standard errors for the estimated parameters. Environmental factors made a substantial contribution to the phenotypic variation, which was overall high for all traits. CONCLUSIONS: There is potential for genetic improvement in all studied traits and estimates of genetic correlations indicate a partly shared genetic architecture among the traits. All estimates have large standard errors. Implementing high-throughput phenotyping is imperative for the estimation of genetic parameters in fast developing insects, and facilitates age synchronisation, which is vital in a breeding population. In spite of endeavours to minimise non-genetic sources of variation, all traits demonstrated substantial influences from environmental components. This emphasises the necessity of thorough attention to the experimental design before breeding is initiated in insect populations.

Temporal regulation of temperature tolerances and gene expression in an arctic insect.

Terrestrial arthropods in the Arctic are exposed to highly variable temperatures that frequently reach cold and warm extremes. Yet, ecophysiological studies on arctic insects typically focus on the ability of species to tolerate low temperatures, whereas studies investigating physiological adaptations of species to periodically warm and variable temperatures are few. In this study, we investigated temporal changes in thermal tolerances and the transcriptome in the Greenlandic seed bug Nysius groenlandicus, collected in the field across different times and temperatures in Southern Greenland. We found that plastic changes in heat and cold tolerances occurred rapidly (within hours) and at a daily scale in the field, and that these changes are correlated with diurnal temperature variation. Using RNA sequencing, we provide molecular underpinnings of the rapid adjustments in thermal tolerance across ambient field temperatures and in the laboratory. We show that transcriptional responses are sensitive to daily temperature changes, and days characterized by high temperature variation induced markedly different expression patterns than thermally stable days. Further, genes associated with laboratory-induced heat responses, including expression of heat shock proteins and vitellogenins, were shared across laboratory and field experiments, but induced at time points associated with lower temperatures in the field. Cold stress responses were not manifested at the transcriptomic level.

Can reintroduction of beavers improve insect biodiversity?

Ecosystem engineering species, such as beavers, may help the restoration of biodiversity. Through the building of dams and lodges and altering the natural hydrology, beavers change the habitat structure and create multiple habitats that facilitate a wide variety of other organisms including terrestrial invertebrate communities. Here we study the effect of beaver reintroduction in Klosterheden in Denmark on biomass of flying invertebrates and diversity of moths. Further, aerial photos were used to assess riparian structure and productivity using the normalized difference vegetation index (NDVI). Our findings show that the presence of beavers affected flying invertebrate biomass, but that this was dependent on time of the year. Further, a strong effect of presence of beavers was found on diversity of moths. The results also show an increase in vegetation productivity and structural heterogeneity at sites with presence of beavers. Overall, our results demonstrate the importance of beavers as important ecosystem engineers that affect invertebrate species composition and abundance, as well as riparian structure and productivity.

Responses of terrestrial polar arthropods to high and increasing temperatures.

Terrestrial arthropods in the Arctic and Antarctic are exposed to extreme and variable temperatures, and climate change is predicted to be especially pronounced in these regions. Available ecophysiological studies on terrestrial ectotherms from the Arctic and Antarctic typically focus on the ability of species to tolerate the extreme low temperatures that can occur in these regions, whereas studies investigating species plasticity and the importance of evolutionary adaptation to periodically high and increasing temperatures are limited. Here, we provide an overview of current knowledge on thermal adaptation to high temperatures of terrestrial arthropods in Arctic and Antarctic regions. Firstly, we summarize the literature on heat tolerance for terrestrial arthropods in these regions, and discuss variation in heat tolerance across species, habitats and polar regions. Secondly, we discuss the potential for species to cope with increasing and more variable temperatures through thermal plasticity and evolutionary adaptation. Thirdly, we summarize our current knowledge of the underlying physiological adjustments to heat stress in arthropods from polar regions. It is clear that very little data are available on the heat tolerance of arthropods in polar regions, but that large variation in arthropod thermal tolerance exists across polar regions, habitats and species. Further, the species investigated show unique physiological adjustments to heat stress, such as their ability to respond quickly to increasing or extreme temperatures. To understand the consequences of climate change on terrestrial arthropods in polar regions, we suggest that more studies on the ability of species to cope with stressful high and variable temperatures are needed.

Responses of terrestrial polar arthropods to high and increasing temperatures.

Terrestrial arthropods in the Arctic and Antarctic are exposed to extreme and variable temperatures, and climate change is predicted to be especially pronounced in these regions. Available ecophysiological studies on terrestrial ectotherms from the Arctic and Antarctic typically focus on the ability of species to tolerate the extreme low temperatures that can occur in these regions, whereas studies investigating species plasticity and the importance of evolutionary adaptation to periodically high and increasing temperatures are limited. Here, we provide an overview of current knowledge on thermal adaptation to high temperatures of terrestrial arthropods in Arctic and Antarctic regions. Firstly, we summarize the literature on heat tolerance for terrestrial arthropods in these regions, and discuss variation in heat tolerance across species, habitats and polar regions. Secondly, we discuss the potential for species to cope with increasing and more variable temperatures through thermal plasticity and evolutionary adaptation. Thirdly, we summarize our current knowledge of the underlying physiological adjustments to heat stress in arthropods from polar regions. It is clear that very little data are available on the heat tolerance of arthropods in polar regions, but that large variation in arthropod thermal tolerance exists across polar regions, habitats and species. Further, the species investigated show unique physiological adjustments to heat stress, such as their ability to respond quickly to increasing or extreme temperatures. To understand the consequences of climate change on terrestrial arthropods in polar regions, we suggest that more studies on the ability of species to cope with stressful high and variable temperatures are needed.

Can reed harvest be used as a management strategy for improving invertebrate biomass and diversity?

The succession-driven reed bed habitat hosts a unique flora and fauna including several endangered invertebrate species. Reed beds can be managed through commercial winter harvest, with implications for reed bed conservation. However, the effects of winter harvest on the invertebrate community are not well understood and vary across studies and taxonomic levels. The aim of this study was to investigate the effects of reed harvest on invertebrate communities. Ground-dwelling and aerial invertebrates were continuously sampled for 10 weeks in the largest coherent reed bed of Scandinavia in order to assess how time since last reed harvest (0, 3, and 25-years) influences invertebrate biomass, biodiversity and community structure across taxonomic levels. Biomass was measured and all specimens were sorted to order level, and Coleoptera was even sorted to species level. The invertebrate community showed distinct compositional differences across the three reed bed ages. Furthermore, biomass of both aerial and ground-dwelling invertebrates was highest in the age-0 reed bed and lowest in the age-25 reed bed. Generally, biodiversity showed an opposite trend with the highest richness and diversity in the age-25 reed bed. We conclude that it is possible to ensure high insect biomass and diversity by creating a mosaic of reed bed of different ages through small-scale harvest in the largest coherent reed bed in Scandinavia. The youngest red beds support a high invertebrate biomass whereas the oldest reed beds support a high biodiversity. Collectively, this elevate our understanding of reed harvest and the effects it has on the invertebrate communities, and might aid in future reed bed management and restoration.

Integrated genome-wide investigations of the housefly, a global vector of diseases reveal unique dispersal patterns and bacterial communities across farms.

BACKGROUND: Houseflies (Musca domestica L.) live in intimate association with numerous microorganisms and is a vector of human pathogens. In temperate areas, houseflies will overwinter in environments constructed by humans and recolonize surrounding areas in early summer. However, the dispersal patterns and associated bacteria across season and location are unclear. We used genotyping-by-sequencing (GBS) for the simultaneous identification and genotyping of thousands of Single Nucleotide Polymorphisms (SNPs) to establish dispersal patterns of houseflies across farms. Secondly, we used 16S rRNA gene amplicon sequencing to establish the variation and association between bacterial communities and the housefly across farms. RESULTS: Using GBS we identified 18,000 SNPs across 400 individuals sampled within and between 11 dairy farms in Denmark. There was evidence for sub-structuring of Danish housefly populations and with genetic structure that differed across season and sex. Further, there was a strong isolation by distance (IBD) effect, but with large variation suggesting that other hidden geographic barriers are important. Large individual variations were observed in the community structure of the microbiome and it was found to be dependent on location, sex, and collection time. Furthermore, the relative prevalence of putative pathogens was highly dependent on location and collection time. CONCLUSION: We were able to identify SNPs for the determination of the spatiotemporal housefly genetic structure, and to establish the variation and association between bacterial communities and the housefly across farms using novel next-generation sequencing (NGS) techniques. These results are important for disease prevention given the fine-scale population structure and IBD for the housefly, and that individual houseflies carry location specific bacteria including putative pathogens.

Heat hardening capacity in Drosophila melanogaster is life stage-specific and juveniles show the highest plasticity.

Variations in stress resistance and adaptive plastic responses during ontogeny have rarely been addressed, despite the possibility that differences between life stages can affect species' range margins and thermal tolerance. Here, we assessed the thermal sensitivity and hardening capacity of Drosophila melanogaster across developmental stages from larval to the adult stage. We observed strong differences between life stages in heat resistance, with adults being most heat resistant followed by puparia, pupae and larvae. The impact of heat hardening (1 h at 35°C) on heat resistance changed during ontogeny, with the highest positive effect of hardening observed in puparia and pupae and the lowest in adults. These results suggest that immobile life stages ( puparia and pupae) have evolved high plasticity in upper thermal limits whereas adults and larvae rely more on behavioural responses to heat stress allowing them to escape from extreme high temperatures. While most studies on the plasticity of heat resistance in ectotherms have focused on the adult life stage, our findings emphasize the crucial importance of juvenile life stages of arthropods in understanding the thermal biology and life stage-specific physiological responses to variable and stressful high temperatures. Failure to acknowledge this complication might lead to biased estimates of species' ability to cope with environmental changes, such as climate change.

Rapid induction of the heat hardening response in an Arctic insect.

The ability to cope with increasing and more variable temperatures, due to predicted climate changes, through plastic and/or evolutionary responses will be crucial for the persistence of Arctic species. Here, we investigate plasticity of heat tolerance of the Greenlandic seed bug Nysius groenlandicus, which inhabits areas with widely fluctuating temperatures. We test the heat tolerance and hardening capacity (plasticity) of N. groenlandicus using both static (heat knock down time, HKDT) and dynamic (critical thermal maximum, CTmax) assays. We find that N. groenlandicus is able to tolerate short-term exposure to temperatures up to almost 50°C and that it can quickly increase heat resistance following heat hardening. Furthermore, we find that this hardening response is reversible within hours after hardening. These findings contrast with common observations from temperate and tropical insects and suggest high thermal plasticity in some Arctic insects which enables them to cope with extreme temperature variability in their habitats.

Automated swimming activity monitor for examining temporal patterns of toxicant effects on individual Daphnia magna.

Aquatic pollutants are often biologically active at low concentrations and impact on biota in combination with other abiotic stressors. Traditional toxicity tests may not detect these effects, and there is a need for sensitive high-throughput methods for detecting sublethal effects. We have evaluated an automated infra-red (IR) light-based monitor for recording the swimming activity of Daphnia magna to establish temporal patterns of toxicant effects on an individual level. Activity was recorded for 48 h and the sensitivity of the monitor was evaluated by exposing D. magna to the reference chemicals K2 Cr2 O7 at 15, 20 and 25 °C and 2,4-dichlorophenol at 20 °C. Significant effects (P < 0.001) of toxicant concentrations, exposure time and incubation temperatures were observed. At 15 °C, the swimming activity remained unchanged for 48 h at sublethal concentrations of K2 Cr2 O7 whereas activity at 20 and 25 °C was more biphasic with decreases in activity occurring after 12-18 h. A similar biphasic pattern was observed after 2,4-dichlorophenol exposure at 20 °C. EC50 values for 2,4-dichlorophenol and K2 Cr2 O7 determined from automated recording of swimming activity showed increasing toxicity with time corresponding to decreases in EC50 of 0.03-0.07 mg l(-1) h(-1) . EC50 values determined after 48 h were comparable or lower than EC50 values based on visual inspection according to ISO 6341. The results demonstrated that the swimming activity monitor is capable of detecting sublethal behavioural effects that are toxicant and temperature dependent. The method allows EC values to be established at different time points and can serve as a high-throughput screening tool in toxicity testing. Copyright © 2015 John Wiley & Sons, Ltd.

Reproductive output and other adult life-history traits of black soldier flies grown on different organic waste and by-products.

The interest in mass-rearing black soldier fly (Hermetia illucens) larvae for food and feed is rapidly increasing. This is partly sparked by the ability of the larvae to efficiently valorise a wide range of organic waste and by-products. Primarily, research has focused on the larval stage, hence underprioritizing aspects of the adult biology, and knowledge on reproduction-related traits such as egg production is needed. We investigated the impact of different organic waste and by-products as larval diets on various life-history traits of adult black soldier flies in a large-scale experimental setup. We reared larvae on four different diets: spent Brewer's grain, ground carrots, Gainesville diet, and ground oranges. Traits assessed were development time to pupa and adult life-stages, adult body mass, female lifespan, egg production, and egg hatch. Larval diet significantly impacted development time to pupa and adult, lifespan, body size, and egg production. In general, flies reared on Brewer's grain developed up to 4.7 d faster, lived up to 2.3 d longer, and produced up to 57% more eggs compared to flies reared on oranges on which they performed worst for these traits. There was no effect of diet type on egg hatch, suggesting that low-nutritious diets, i.e. carrots and oranges, do not reduce the quality but merely the quantity of eggs. Our results demonstrate the importance of larval diet on reproductive output and other adult traits, all important for an efficient valorisation of organic waste and by-products, which is important for a sustainable insect-based food and feed production.

Framework for valorizing waste- and by-products through insects and their microbiomes for food and feed.

One third of the food produced for human consumption is currently lost or wasted. Insects have a high potential for converting organic waste- and by-products into food and feed for a growing human population due to symbiosis with microorganisms. These symbioses provide an untapped reservoir of functional microbiomes that can be used to improve industrial insect production but are poorly studied in most insect species. Here we review the most current understanding and challenges of valorizing organic waste- and by-products through insects and their microbiomes for food and feed, and emerging novel food technologies that can be used to investigate and manipulate host(insects)-microbiome interactions. We further construct a holistic framework, by integration of novel food technologies including holo-omics, genome editing, breeding, phage therapy, and administration of prebiotics and probiotics to investigate and manipulate host(insects)-microbiome interactions, and solutions for achieving stakeholder acceptance of novel food technologies for a sustainable food production.

Foodborne disease prevention and broiler chickens with reduced Campylobacter infection.

Studies have suggested that flies play a linking role in the epidemiology of Campylobacter spp. in broiler chickens and that fly screens can reduce the prevalence of Campylobacter spp. We examined the year-round and long-term effects of fly screens in 10 broiler chicken houses (99 flocks) in Denmark. Prevalence of Campylobacter spp.-positive flocks was significantly reduced, from 41.4% during 2003-2005 (before fly screens) to 10.3% in 2006-2009 (with fly screens). In fly screen houses, Campylobacter spp. prevalence did not peak during the summer. Nationally, prevalence of Campylobacter spp.-positive flocks in Denmark could have been reduced by an estimated 77% during summer had fly screens been part of biosecurity practices. These results imply that fly screens might help reduce prevalence of campylobacteriosis among humans, which is closely linked to Campylobacter spp. prevalence among broiler chicken flocks.

Into the wild-a field study on the evolutionary and ecological importance of thermal plasticity in ectotherms across temperate and tropical regions.

Understanding how environmental factors affect the thermal tolerance of species is crucial for predicting the impact of thermal stress on species abundance and distribution. To date, species' responses to thermal stress are typically assessed on laboratory-reared individuals and using coarse, low-resolution, climate data that may not reflect microhabitat dynamics at a relevant scale. Here, we examine the daily temporal variation in heat tolerance in a range of species in their natural environments across temperate and tropical Australia. Individuals were collected in their habitats throughout the day and tested for heat tolerance immediately thereafter, while local microclimates were recorded at the collection sites. We found high levels of plasticity in heat tolerance across all the tested species. Both short- and long-term variability of temperature and humidity affected plastic adjustments of heat tolerance within and across days, but with species differences. Our results reveal that plastic changes in heat tolerance occur rapidly at a daily scale and that environmental factors on a relatively short timescale are important drivers of the observed variation in thermal tolerance. Ignoring such fine-scale physiological processes in distribution models might obscure conclusions about species' range shifts with global climate change. This article is part of the theme issue 'Species' ranges in the face of changing environments (part 1)'.

Rapid Adjustments in Thermal Tolerance and the Metabolome to Daily Environmental Changes - A Field Study on the Arctic Seed Bug Nysius groenlandicus.

Laboratory investigations on terrestrial model-species, typically of temperate origin, have demonstrated that terrestrial ectotherms can cope with daily temperature variations through rapid hardening responses. However, few studies have investigated this ability and its physiological basis in the field. Especially in polar regions, where the temporal and spatial temperature variations can be extreme, are hardening responses expected to be important. Here, we examined diurnal adjustments in heat and cold tolerance in the Greenlandic seed bug Nysius groenlandicus by collecting individuals for thermal assessment at different time points within and across days. We found a significant correlation between observed heat or cold tolerance and the ambient microhabitat temperatures at the time of capture, indicating that N. groenlandicus continuously and within short time-windows respond physiologically to thermal changes and/or other environmental variables in their microhabitats. Secondly, we assessed underlying metabolomic fingerprints using GC-MS metabolomics in a subset of individuals collected during days with either low or high temperature variation. Concentrations of metabolites, including sugars, polyols, and free amino acids varied significantly with time of collection. For instance, we detected elevated sugar levels in animals caught at the lowest daily field temperatures. Polyol concentrations were lower in individuals collected in the morning and evening and higher at midday and afternoon, possibly reflecting changes in temperature. Additionally, changes in concentrations of metabolites associated with energetic metabolism were observed across collection times. Our findings suggest that in these extreme polar environments hardening responses are marked and likely play a crucial role for coping with microhabitat temperature variation on a daily scale, and that metabolite levels are actively altered on a daily basis.

Growth and metabolic performance of black soldier fly larvae grown on low and high-quality substrates.

We have measured growth and respiration in black soldier fly (BSF) larvae fed with mixtures of a low-quality substrate (degassed sludge, DS), and a high-quality substrate (chicken feed, CF) in order to elucidate how substrate quality affect larval metabolism and feed conversion into new biomass. The BSF larvae grew faster and became larger the higher the content of CF was in the substrate. Growth followed a sigmoidal curve from where the specific growth rate was determined and compared to measured specific respiration rates, in order to estimate costs of growth, maintenance metabolism, rates of feed assimilation, and net growth efficiency. Specific feed assimilation rates were similar on all substrate mixtures. Maximal specific growth rates were also affected only little unless the larvae were grown in pure DS. In contrast, the cost of growth and the maintenance metabolism were larger the higher the proportion of DS was. High specific growth rates were, in addition, sustained for shorter periods of the time the more DS was included in the substrate mixtures. In effect, higher proportions of the assimilated feed were spent on respiratory purposes instead of being converted into larval biomass and the net growth efficiency decreased the more DS was included in the substrate mixtures. We conclude that substrate quality may affect the conversion of feed into new biomass via alterations of the metabolic performance of BSF larvae and thereby the overall performance of BSF larval cultures.

Strong isolation by distance among local populations of an endangered butterfly species (Euphydryas aurinia).

The marsh fritillary (Euphydryas aurinia) is a critically endangered butterfly species in Denmark known to be particularly vulnerable to habitat fragmentation due to its poor dispersal capacity. We identified and genotyped 318 novel SNP loci across 273 individuals obtained from 10 small and fragmented populations in Denmark using a genotyping-by-sequencing (GBS) approach to investigate its population genetic structure. Our results showed clear genetic substructuring and highly significant population differentiation based on genetic divergence (F ST) among the 10 populations. The populations clustered in three overall clusters, and due to further substructuring among these, it was possible to clearly distinguish six clusters in total. We found highly significant deviations from Hardy-Weinberg equilibrium due to heterozygote deficiency within every population investigated, which indicates substructuring and/or inbreeding (due to mating among closely related individuals). The stringent filtering procedure that we have applied to our genotype quality could have overestimated the heterozygote deficiency and the degree of substructuring of our clusters but is allowing relative comparisons of the genetic parameters among clusters. Genetic divergence increased significantly with geographic distance, suggesting limited gene flow at spatial scales comparable to the dispersal distance of individual butterflies and strong isolation by distance. Altogether, our results clearly indicate that the marsh fritillary populations are genetically isolated. Further, our results highlight that the relevant spatial scale for conservation of rare, low mobile species may be smaller than previously anticipated.