Target and non-target effects of insecticide use during ornamental milkweed production.

There are widespread public efforts to conserve wildlife in urbanized landscapes via the installation of nursery-grown plants that support Lepidoptera taxa. Insecticides are commonly used during nursery production to suppress key plant pests, and many products have extended periods of toxicity and affect a wide range of herbivore taxa. While there are plentiful toxicological data on bee species, predominantly the Western honey bee (Apis mellifera L.), little is known about how insecticides affect nonpest lepidopterans. Lepidoptera has different modes of exposure (e.g., leaf-feeding) and differences in susceptibility to insecticide target sites compared to bees. Consequently, many products compatible with bee conservation pose an uncertain risk to nonpest lepidopterans and thus may represent an under-recognized conflict with conservation efforts. Using the monarch butterfly (Danaus plexippus, L.), tropical milkweed (Asclepias curassavica, L.), and oleander aphid (Aphis nerii, Fonscolombe, 1841) system, we conducted leaf and whole-plant feeding assays to evaluate effects of acute and chronic monarch exposure to industry standard and alternative reduced-risk insecticides used during nursery production. We also evaluated the efficacy of these insecticides against their target pest, the oleander aphid. Our results indicate that insecticides used to control pests on ornamental milkweed can cause monarch larval mortality up to 4 wk after treatment application. Furthermore, the duration of aphid suppression is often shorter than the duration of adverse effects on monarchs. This study demonstrates a conflict between insect pest management and Lepidoptera conservation during ornamental plant production and has implications for the conservation value of ornamentals after retail sale.

Aphid infestations reduce monarch butterfly colonization, herbivory, and growth on ornamental milkweed.

Anthropogenic disturbance is driving global biodiversity loss, including the monarch butterfly (Danaus plexippus), a dietary specialist of milkweed. In response, ornamental milkweed plantings are increasingly common in urbanized landscapes, and recent evidence indicates they have conservation value for monarch butterflies. Unfortunately, sap-feeding insect herbivores, including the oleander aphid (Aphis nerii), frequently reach high densities on plants in nursery settings and urbanized landscapes. Aphid-infested milkweed may inhibit monarch conservation efforts by reducing host plant quality and inducing plant defenses. To test this, we evaluated the effects of oleander aphid infestation on monarch oviposition, larval performance, and plant traits using tropical milkweed (Asclepias curassavica), the most common commercially available milkweed species in the southern U.S. We quantified monarch oviposition preference, larval herbivory, larval weight, and plant characteristics on aphid-free and aphid-infested milkweed. Monarch butterflies deposited three times more eggs on aphid-free versus aphid-infested milkweed. Similarly, larvae fed aphid-free milkweed consumed and weighed twice as much as larvae fed aphid-infested milkweed. Aphid-free milkweed had higher total dry leaf biomass and nitrogen content than aphid-infested milkweed. Our results indicate that oleander aphid infestations can have indirect negative impacts on urban monarch conservation efforts and highlight the need for effective Lepidoptera-friendly integrated pest management tactics for ornamental plants.

Protective Benefits of Tending Ants to a Critically Endangered Butterfly.

Ants provide protection to various organisms via myrmecophilous relationships. Most notably, ants and several butterfly species are involved in mainly mutualistic interactions. Previous field studies have shown that butterfly larval survival is increased in the presence of tending ants, suggesting that ants are providing protection against insect predation or parasitism. Here, we conducted a series of timed observational trials under laboratory conditions to assess larval survival and ant protection from insect predators for a myrmecophilous lycaenid butterfly. We focused on a critically endangered butterfly, the Miami blue (Cyclargus thomasi bethunebakeri) (Comstock and Huntington) (Lepidoptera: Lycaenidae), and its most common ant associate, the Florida carpenter ant (Camponotus floridanus) (Buckley) (Hymenoptera: Formicidae), to test this assumption of ant protection. We found that ants provide significant protection to Miami blue larvae, with later instar larvae receiving a higher level of protection due to differences in tending frequencies. These results will aid in informing conservation management and future organism reintroductions for this endangered butterfly.

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations.

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address existing knowledge gaps. This protocol represents a demonstrated method for nondestructively sampling rare butterflies for population genetic structure or DNA barcoding analyses. It uses the chorion of hatched butterfly ovae to yield sufficiently high quantity and quality DNA for successful gene sequencing to confirm species identity and quantify genetic variation. It may be particularly useful when other tissue sampling techniques are impractical or unavailable. While developed for a lepidopteran, it nonetheless could easily be adapted for use with other insect species. It was specifically designed with ease of use as a goal to help maximize broad implementation by individuals of varying experience and skill levels, such as community scientists, conservation practitioners, and students, and for use over large geographic areas to facilitate broad population sampling. The data generated can help inform taxonomic and listing decisions, conservation and management actions, and enhance basic ecological research.

Climate change effects on animal ecology: butterflies and moths as a case study.

Butterflies and moths (Lepidoptera) are one of the most studied, diverse, and widespread animal groups, making them an ideal model for climate change research. They are a particularly informative model for studying the effects of climate change on species ecology because they are ectotherms that thermoregulate with a suite of physiological, behavioural, and phenotypic traits. While some species have been negatively impacted by climatic disturbances, others have prospered, largely in accordance with their diversity in life-history traits. Here we take advantage of a large repertoire of studies on butterflies and moths to provide a review of the many ways in which climate change is impacting insects, animals, and ecosystems. By studying these climate-based impacts on ecological processes of Lepidoptera, we propose appropriate strategies for species conservation and habitat management broadly across animals.

Bloom evenness modulates the influence of bloom abundance on insect community structure in suburban gardens.

As land use change drives global insect declines, the value of enhancing habitat in urban and suburban landscapes has become increasingly important for flower-visiting insects. In order to help identify best landscaping practices, we conducted plant surveys and insect bowl-trap surveys in 34 suburban yards for 21 months in Gainesville, FL, USA, which resulted in 274 paired days of plant and insect survey data. We assessed the impact of nearest greenspace size, distance to greenspace, yard area, plant richness, plant type, bloom abundance, bloom richness and bloom evenness on insect abundance and richness. Our samples include 34,972 insects captured, 485,827 blooms counted and 774 species of plants recorded. We found that bloom evenness had a modulating effect on bloom abundance-a more even sample of the same number of blooms would have a disproportionately greater positive impact on flower visitor richness, insect richness and insect abundance. Bloom abundance was also highly significant and positively associated with flower visitor abundance, but nearest greenspace size, distance to greenspace, plant type (native vs. non-native vs. Florida Friendly), and yard area were not found to be important factors. Plant richness was a highly significant factor, but its effect size was very small.

Mixed-Species Gardens Increase Monarch Oviposition without Increasing Top-Down Predation.

Monarch butterfly populations have declined by over 80% in the last 20 years. Conservation efforts focus on the creation of milkweed habitats to mitigate this decline. Previous research has found monarchs lay more eggs per milkweed stem in urban gardens than natural habitats and recent work identified specific garden designs that make urban gardens more attractive to monarchs. Increasing plant diversity can reduce specialist insect herbivore colonization via bottom-up (e.g., plant) and top-down (e.g., predation) regulatory factors. Although this is beneficial for pest management efforts, it contradicts conservation efforts. In this study, we explored if adding multiple flowering species to garden-sized milkweed plantings affected monarch oviposition or top-down regulation of larvae. We compared monarch egg abundance, natural enemy abundance and richness, and biological control of monarch larvae in milkweed monocultures and milkweed mixed with four additional wildflower species. We found that monarchs laid 22% more eggs on sentinel milkweed plants in mixed-species plots with no effect of plant diversity on monarch survival. We also found higher natural enemy richness, wasp, and predatory bug abundance in the mixed-species plots and this did not translate to higher biological control rates. Our results provide more evidence that plant selection and habitat design are important for monarch conservation.

Uptake and toxicity of clothianidin to monarch butterflies from milkweed consumption.

Recent concern for the adverse effects from neonicotinoid insecticides has centered on risk for insect pollinators in general and bees specifically. However, natural resource managers are also concerned about the risk of neonicotinoids to conservation efforts for the monarch butterfly (Danaus plexippus) and need additional data to help estimate risk for wild monarch butterflies exposed to those insecticides. In the present study, monarch butterfly larvae were exposed in the laboratory to clothianidin via contaminated milkweed plants from hatch until pupation, and the effects upon larval survival, larval growth, pupation success, and adult size were measured. Soils dosed with a granular insecticide product led to mean clothianidin concentrations of 10.8-2,193 ng/g in milkweed leaves and 5.8-58.0 ng/g in larvae. Treatment of soils also led to clothianidin concentrations of 2.6-5.1 ng/g in adult butterflies indicating potential for transfer of systemic insecticides from the soil through plants and larvae to adult butterflies. Estimated LC50s for total mortality (combined mortality of larvae and pupae) and EC50 for larval growth were variable but higher than the majority of concentrations reported in the literature for clothianidin contamination of leaves.

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques.

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Phylogeography and population genetics of pine butterflies: Sky islands increase genetic divergence.

The sky islands of southeastern Arizona (AZ) mark a major transition zone between tropical and temperate biota and are considered a neglected biodiversity hotspot. Dispersal ability and host plant specificity are thought to impact the history and diversity of insect populations across the sky islands. We aimed to investigate the population structure and phylogeography of two pine-feeding pierid butterflies, the pine white (Neophasia menapia) and the Mexican pine white (Neophasia terlooii), restricted to these "islands" at this transition zone. Given their dependence on pines as the larval hosts, we hypothesized that habitat connectivity affects population structure and is at least in part responsible for their allopatry. We sampled DNA from freshly collected butterflies from 17 sites in the sky islands and adjacent high-elevation habitats and sequenced these samples using ddRADSeq. Up to 15,399 SNPs were discovered and analyzed in population genetic and phylogenetic contexts with Stacks and pyRAD pipelines. Low genetic differentiation in N. menapia suggests that it is panmictic. Conversely, there is strong evidence for population structure within N. terlooii. Each sky island likely contains a population of N. terlooii, and clustering is hierarchical, with populations on proximal mountains being more related to each other. The N. menapia habitat, which is largely contiguous, facilitates panmixia, while the N. terlooii habitat, restricted to the higher elevations on each sky island, creates distinct population structure. Phylogenetic results corroborate those from population genetic analyses. The historical climate-driven fluxes in forest habitat connectivity have implications for understanding the biodiversity of fragmented habitats.

Drifting Phenologies Cause Reduced Seasonality of Butterflies in Response to Increasing Temperatures.

Climate change has caused many ecological changes around the world. Altered phenology is among the most commonly observed effects of climate change, and the list of species interactions affected by altered phenology is growing. Although many studies on altered phenology focus on single species or on pairwise species interactions, most ecological communities are comprised of numerous, ecologically similar species within trophic groups. Using a 12-year butterfly monitoring citizen science data set, we aimed to assess the degree to which butterfly communities may be changing over time. Specifically, we wanted to assess the degree to which phenological sensitivities to temperature could affect temporal overlap among species within communities, independent of changes in abundance, species richness, and evenness. We found that warming winter temperatures may be associated with some butterfly species making use of the coldest months of the year to fly as adults, thus changing temporal co-occurrence with other butterfly species. Our results suggest that changing temperatures could cause immediate restructuring of communities without requiring changes in overall abundance or diversity. Such changes could have fitness consequences for individuals within trophic levels by altering competition for resources, as well as indirect effects mediated by species interactions across trophic levels.

Evaluating an Alleged Mimic of the Monarch Butterfly: Neophasia (Lepidoptera: Pieridae) Butterflies are Palatable to Avian Predators.

Some taxa have adopted the strategy of mimicry to protect themselves from predation. Butterflies are some of the best representatives used to study mimicry, with the monarch butterfly, Danaus plexippus (Lepidoptera: Nymphalidae) a well-known model. We are the first to empirically investigate a proposed mimic of the monarch butterfly: Neophasia terlooii, the Mexican pine white butterfly (Lepidoptera: Pieridae). We used captive birds to assess the palatability of N. terlooii and its sister species, N. menapia, to determine the mimicry category that would best fit this system. The birds readily consumed both species of Neophasia and a palatable control species but refused to eat unpalatable butterflies such as D. plexippus and Heliconius charithonia (Lepidoptera: Nymphalidae). Given some evidence for mild unpalatability of Neophasia, we discuss the results considering modifications to classic mimicry theory, i.e., a palatability-based continuum between Batesian and Müllerian mimicry, with a quasi-Batesian intermediate. Understanding the ecology of Neophasia in light of contemporary and historical sympatry with D. plexippus could shed light on the biogeography of, evolution of, and predation pressure on the monarch butterfly, whose migration event has become a conservation priority.

Behavioral Evidence for Host Transitions in Plant, Plant Parasite, and Insect Interactions.

Specialized herbivorous insects have the ability to transition between host plant taxa, and considering the co-evolutionary history between plants and the organisms utilizing them is important to understanding plant insect interactions. We investigated the role of a pine tree parasite, dwarf mistletoe (Arceuthobium spp.) M. Bieb. Santalales: Viscaceae, in mediating interactions between Neophasia (Lepidoptera: Pieridae) butterflies and pine trees, the butterflies' larval hosts. Mistletoe is considered the butterflies' ancestral host, and the evolutionary transition to pine may have occurred recently. In Arizona, United States, we studied six sites in pine forest habitats: three in Neophasia menapia (Felder and R. Felder, 1859) habitat and three in Neophasia terlooii Behr, 1869 habitat. Each site contained six stands of trees that varied in mistletoe infection severity. Butterfly behavior was observed and ranked at each stand. Volatile compounds were collected from trees at each site and analyzed using gas chromatography-mass spectroscopy. Female butterflies landed on or patrolled around pine trees (i.e., interacted) more than males, and N. terlooii interacted more with pine trees than N. menapia. Both butterfly species interacted more with tree stands harboring greater mistletoe infection, and N. terlooii interacted more with heavily infected tree stands than did N. menapia. The influence of mistletoe on Neophasia behavior may be mediated by differences in tree volatiles resulting from mistletoe infection. Volatile profiles significantly differed between infected and uninfected pine trees. The role of mistletoe in mediating butterfly interactions with pines has implications for conservation biology and forest management, and highlights the importance of understanding an organism's niche in an evolutionary context.

Differences in winter cold hardiness reflect the geographic range disjunction of Neophasia menapia and Neophasia terlooii (Lepidoptera: Pieridae).

Predicting how rapid climate change will affect terrestrial biota depends on a thorough understanding of an organism's biology and evolutionary history. Organisms at their range boundaries are particularly sensitive to climate change. As predominantly terrestrial poikilotherms, insects are often geographically limited by extremes in ambient temperatures. We compared the cold hardiness strategies of two geographically widespread butterflies, the pine white, Neophasia menapia, and the Mexican pine white, N. terlooii (Lepidoptera: Pieridae), at the near-contact zone of their range boundaries. Eggs are laid on pine needles and are exposed to harsh winter conditions. Eggs were collected from wild-caught butterflies, and we determined the supercooling point (SCP) and lower lethal temperature (LLT50) of overwintering eggs. The SCP of Neophasia menapia eggs (-29.0 ±â€¯0.6 °C) was significantly lower than that of N. terlooii eggs (-21.8 ±â€¯0.7 °C). Both species were freeze-intolerant and capable of surviving down to their respective SCPs (LLT50 of N. menapia between -30 and -31 °C, N. terlooii between -20 and -21 °C). Cold exposure time did not affect the survival of N. menapia, but N. terlooii experienced somewhat greater mortality at sub-freezing temperatures during longer exposures. Our results, coupled with an analysis of microclimate data, indicate that colder winters in northern Arizona may contribute to the northern range limit for N. terlooii. Furthermore, careful analysis of historical weather data indicates that mortality from freezing is unlikely in southern Arizona but possible in northern Arizona. Movements of Neophasia range boundaries could be monitored as potential biological responses to climate change.

Fruit Set and Single Visit Stigma Pollen Deposition by Managed Bumble Bees and Wild Bees in Citrullus lanatus (Cucurbitales: Cucurbitaceae).

Pollinators provide essential services for watermelon, Citrullus lanatus (Thunb.; Cucurbitales: Cucurbitaceae). Managed bumble bees, Bombus impatiens (Cresson; Hymenoptera: Apidae), have been shown to be a useful watermelon pollinator in some areas. However, the exact contribution bumble bees make to watermelon pollination and how their contribution compares to that of other bees is unclear. We used large cages (5.4 × 2.5 × 2.4 m) to confine bumble bee hives to watermelon plants and compared fruit set in those cages to cages containing watermelons but no pollinators, and to open areas of field next to cages (allows all pollinators). We also collected data on single visit pollen deposition onto watermelon stigmas by managed bumble bees, honey bees, and wild bees. Overall, more fruit formed within the open cages than in cages of the other two treatment groups. B. impatiens and Melissodes spp. deposited the most pollen onto watermelon stigmas per visit, but all bee species observed visiting watermelon flowers were capable of depositing ample pollen to watermelon stigmas. Although B. impatiens did deposit large quantities of pollen to stigmas, they were not common within the field (i.e., outside the cages) as they were readily drawn to flowering plants outside of the watermelon field. Overall, bumble bees can successfully pollinate watermelon, but may be useful in greenhouses or high tunnels where watermelon flowers have no competition from other flowering plants that could draw bumble bees away from watermelon.

Trap Nesting Wasps and Bees in Agriculture: A Comparison of Sown Wildflower and Fallow Plots in Florida.

Wildflower strip plantings in intensive agricultural systems have become a widespread tool for promoting pollination services and biological conservation because of their use by wasps and bees. Many of the trap-nesting wasps are important predators of common crop pests, and cavity-nesting bees that utilize trap-nests are important pollinators for native plants and many crops. The impact of wildflower strips on the nesting frequency of trap-nesting wasps or bees within localized areas has not been thoroughly investigated. Trap-nests made of bamboo reeds (Bambusa sp.) were placed adjacent to eight 0.1 ha wildflower plots and paired fallow areas (control plots) to determine if wildflower strips encourage the nesting of wasps and bees. From August 2014 to November 2015, occupied reeds were gathered and adults were collected as they emerged from the trap-nests. Treatment (wildflower or fallow plots) did not impact the number of occupied reeds or species richness of trap-nesting wasps using the occupied reeds. The wasps Pachodynerus erynnis, Euodynerus megaera, Parancistrocerus pedestris, and Isodontia spp. were the most common trap-nesting species collected. Less than 2% of the occupied reeds contained bees, and all were from the genus Megachile. The nesting wasp and bee species demonstrated preferences for reeds with certain inside diameters (IDs). The narrow range of ID preferences exhibited by each bee/wasp may provide opportunities to take advantage of their natural histories for biological control and/or pollination purposes.

Jamaica's Critically Endangered Butterfly: A Review of the Biology and Conservation Status of the Homerus Swallowtail (Papilio (Pterourus) homerus Fabricius).

The Homerus swallowtail, Papilio (Pterourus) homerus Fabricius, is listed as an endangered species and is endemic to the Caribbean island of Jamaica. The largest butterfly in the Western Hemisphere, P. homerus once inhabited seven of Jamaica's 14 parishes and consisted of at least three populations; however, now only two stronghold populations remain, a western population in the rugged Cockpit Country and an eastern population in the Blue and John Crow Mountains. Despite numerous studies of its life history, much about the population biology, including estimates of total numbers of individuals in each population, remains unknown. In addition, a breeding program is needed to establish an experimental population, which could be used to augment wild populations and ensure the continued survival of the species. Here, we present a review of the biology of P. homerus and recommendations for a conservation plan.

Can butterflies evade fire? Pupa location and heat tolerance in fire prone habitats of Florida.

Butterflies such as the atala hairstreak, Eumaeus atala Poey, and the frosted elfin, Callophrys irus Godart, are restricted to frequently disturbed habitats where their larval host plants occur. Pupae of these butterflies are noted to reside at the base of host plants or in the leaf litter and soil, which may allow them to escape direct mortality by fire, a prominent disturbance in many areas they inhabit. The capacity of these species to cope with fire is a critical consideration for land management and conservation strategies in the locations where they are found. Survival of E. atala pupae in relation to temperature and duration of heat pulse was tested using controlled water bath experiments and a series of prescribed fire field experiments. Survival of E. atala pupae was correlated to peak temperature and heat exposure in both laboratory and field trials. In addition, E. atala survival following field trials was correlated to depth of burial; complete mortality was observed for pupae at the soil surface. Fifty percent of E. atala survived the heat generated by prescribed fire when experimentally placed at depths ≥ 1.75 cm, suggesting that pupation of butterflies in the soil at depth can protect from fatal temperatures caused by fire. For a species such as E. atala that pupates above ground, a population reduction from a burn event is a significant loss, and so decreasing the impact of prescribed fire on populations is critical.

Genetic estimates of contemporary effective population size in an endangered butterfly indicate a possible role for genetic compensation.

The effective population size (N e ) is a critical evolutionary and conservation parameter that can indicate the adaptive potential of populations. Robust estimates of N e of endangered taxa have been previously hampered by estimators that are sensitive to sample size. We estimated N e on two remaining populations of the endangered Miami blue butterfly, a formerly widespread taxon in Florida. Our goal was to determine the consistency of various temporal and point estimators on inferring N e and to determine the utility of this information for understanding the role of genetic stochasticity. We found that recently developed 'unbiased estimators' generally performed better than some older methods in that the former had more realistic N e estimates and were more consistent with what is known about adult population size. Overall, N e /N ratios based on census point counts were high. We suggest that this pattern may reflect genetic compensation caused by reduced reproductive variance due to breeding population size not being limited by resources. Assuming N e and N are not heavily biased, it appears that the lack of gene flow between distant populations may be a greater genetic threat in the short term than the loss of heterozygosity due to inbreeding.

Development and characterization of polymorphic microsatellite loci in the endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri).

The Miami blue butterfly (Cyclargus thomasi bethunebakeri) is a state-endangered taxon in Florida and a candidate for federal listing. Here we report 12 polymorphic microsatellite loci appropriate for use in population and conservation studies. We genotyped 114 individuals sampled from a metapopulation in the lower Florida Keys over a 2-year period (2005-2006). These results show 4-14 alleles per locus, and ranges of observed and expected heterozygosities are 0.02679-0.79630 and 0.06154-0.69565, respectively. Large deviations from Hardy-Weinberg equilibrium (HWE) are observed across the whole sample set. When a single breeding population is analysed alone, seven of the loci are in HWE.