Sasakia Charonda (Apaturinae: Nymphalidae) and its Three Related Butterfly Larvae Use Halitosis to Repel Predators

Accidentally, we discovered that Sasakia charonda (Nymphalidae: Apaturinae) larvae disturbed by ants or humans released volatile compounds from their mouth; thus, we tried to identify these halitosis. We collected halitosis directly from the mouths of S. charonda larvae into volatile-collecting tubes. Trapped halitosis were subjected to gas chromatography-mass spectrometry (GC–MS). We confirmed the identity of eleven substances by comparison to GC of known standards, and inferred them to be mainly alcohols and aldehydes/ketones, with main chains of 4–5 carbons. Three of the chemicals in these halitosis, 2-butanol, 1-penten-3-ol and 3-pentanone, affected the behavior of Pristomyrmex punctatus and Formica japonica ants that co-inhabited the S. charonda rearing cage. We concluded that the substances we identified in this study were used as defensive halitosis, analogous to osmeterium emissions specific to Papilionidae butterflies. Based on smell, Holikawa found that Hestina assimilis and H. persimilis larvae have closely related halitosis. Thus, we also analyzed the halitosis of these two species as well as Apatura metis, another Apaturinae, using the same methods. We found that these species also release halitosis. The composition of the substances of H. assimilis and H. persimilis were somewhat similar to that of S. charonda, whereas that of A. metis differed. Some of the substances also induced defensive behavior in these species of Apaturinae larvae.

Ecology and Genetic Structure of the Parasitoid Phobocampe confusa (Hymenoptera: Ichneumonidae) in Relation to Its Hosts, Aglais Species (Lepidoptera: Nymphalidae)

The biology of parasitoids in natural ecosystems remains very poorly studied, though they are key species for their functioning. Here we focused on Phobocampe confusa, a Nymphalini specialist, responsible for high mortality rates in charismatic butterfly species in Europe (genus Aglais). We studied its ecology and genetic structure in connection with those of its host butterflies in Sweden. To this aim, we gathered data from 428 P. confusa individuals reared from 6094 butterfly larvae (of A. urticae, A. io, and in two occasions of Araschnia levana) collected over two years (2017 and 2018) and across 19 sites distributed along a 500 km latitudinal gradient. We found that P. confusa is widely distributed along the latitudinal gradient. Its distribution seems constrained over time by the phenology of its hosts. The large variation in climatic conditions between sampling years explains the decrease in phenological overlap between P. confusa and its hosts in 2018 and the 33.5% decrease in the number of butterfly larvae infected. At least in this study, P. confusa seems to favour A. urticae as host. While it parasitized nests of A. urticae and A. io equally, the proportion of larvae parasitized is significantly higher for A. urticae. At the landscape scale, P. confusa is almost exclusively found in vegetated open land and near deciduous forests, whereas artificial habitats are negatively correlated with the likelihood of a nest to be parasitized. The genetic analyses on 89 adult P. confusa and 87 adult A. urticae using CO1 and AFLP markers reveal a low genetic diversity in P. confusa and a lack of genetic structure in both species, at the scale of our sampling. Further genetic studies using high-resolution genomics tools will be required to better understand the population genetic structure of P. confusa, its biotic interactions with its hosts, and ultimately the stability and the functioning of natural ecosystems.

Ecology and population genetics of the parasitoid Phobocampe confusa (Hymenoptera: Ichneumonidae) in relation to its hosts, Aglais species (Lepidoptera: Numphalidae)

The biology of parasitoids in natural ecosystems remain very poorly studied, while they are key species for their functioning. Here we focused on Phobocampe confusa, a vanessines specialist, responsible for high mortality rates in very emblematic butterfly species in Europe (genus Aglais). We studied its ecology and genetic structure in connection with those of its host butterflies in Sweden. To this aim, we gathered data from 428 P. confusa individuals reared from 6094 butterfly larvae (of A. urticae, A. io and in two occasions of Araschnia levana) collected over two years (2017 and 2018) and 19 sites distributed along a 500 km latitudinal gradient. We found that P. confusa is widely distributed along the latitudinal gradient. Its distribution is constrained over time by the phenology of its hosts. The large variation in climatic conditions between sampling years explains the decrease in phenological overlap between P. confusa and its hosts in 2018 and the 33.5% decrease in the number of butterfly larvae infected. At least in this study, P. confusa seems to favour A. urticae as host: while it parasitized nests of A. urticae and A. io equally, the proportion of larvae is significantly higher for A. urticae. At the landscape scale, P. confusa is almost exclusively found in vegetated open land and near deciduous forests, whereas artificial habitats are negatively correlated with the likelihood of a nest to be parasitized. The genetic analyses on 89 adult P. confusa and 87 adult A. urticae using COI and AFLP markers reveal a low genetic diversity in P. confusa and a lack of population genetic structure in both species, at the scale of our sampling. Further genetic studies using high-resolution genomics tools will be required to better understand the population genetic structure of P. confusa, its biotic interactions with its hosts, and ultimately the stability and the functioning of natural ecosystems.

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.

Disrupting butterfly caterpillar microbiomes does not impact their survival and development

Associations with gut microbes are believed to play crucial roles in the physiology, immune function, development and behaviour of insects. However, microbiome sequencing has recently suggested that butterflies are an anomaly, because their microbiomes do not show strong host- and developmental stage-specific associations. We experimentally manipulated butterfly larval gut microbiota and found that disrupting gut microbes had little influence on larval survival and development. Larvae of the butterflies Danaus chrysippus and Ariadne merione that fed on chemically sterilized or antibiotic-treated host plant leaves had significantly reduced bacterial loads, and their gut bacterial communities were disrupted substantially. However, neither host species treated this way suffered a significant fitness cost: across multiple experimental blocks, treated and control larvae had similar survival, growth and development. Furthermore, re-introducing microbes from the excreta of control larvae did not improve larval growth and survival. Thus, these butterfly larvae did not appear to rely on specialized gut bacteria for digestion, detoxification, biomass accumulation and metamorphosis. Our experiments thus show that dependence on gut bacteria for growth and survival is not a universal phenomenon across insects. Our findings also caution that strategies which target gut microbiomes may not always succeed in controlling Lepidopteran pests.

Tolerance of High Oral Doses of Nonradioactive and Radioactive Caesium Chloride in the Pale Grass Blue Butterfly Zizeeria maha

The biological effects of the Fukushima nuclear accident have been examined in the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae). In previous internal exposure experiments, larvae were given field-collected contaminated host plant leaves that contained up to 43.5 kBq/kg (leaf) of radioactive caesium. Larvae ingested up to 480 kBq/kg (larva), resulting in high mortality and abnormality rates. However, these results need to be compared with the toxicological data of caesium. Here, we examined the toxicity of both nonradioactive and radioactive caesium chloride on the pale grass blue butterfly. Larvae were fed a caesium-containing artificial diet, ingesting up to 149 MBq/kg (larva) of radioactive caesium (137Cs) or a much higher amount of nonradioactive caesium. We examined the pupation rate, eclosion rate, survival rate up to the adult stage, and the forewing size. In contrast to previous internal exposure experiments using field-collected contaminated leaves, we could not detect any effect. We conclude that the butterfly is tolerant to ionising radiation from 137Cs in the range tested but is vulnerable to radioactive contamination in the field. These results suggest that the biological effects in the field may be mediated through ecological systems and cannot be estimated solely based on radiation doses.

Varied Effects of Tending Ant Species on the Development of Facultatively Myrmecophilous Lycaenid Butterfly Larvae

Ants often tend and protect the larvae of various myrmecophilous lycaenid species, which influences the fitness of butterflies by altering their growth and developmental time. Tending produces diverse effects depending on lycaenid sex and the lycaenid/ant species combination. Effects are widely variable, especially in facultatively myrmecophilous lycaenids such as Plebejus argyrognomon praeterinsularis, because they are associated with several ant species and can survive without any ant tending. We studied the effects of ant tending on the adult body mass and larval developmental time of P. argyrognomon praeterinsularis. Female larvae grew significantly heavier as adults when tended by Camponotus japonicus rather than by either Lasius japonicus or no ant species. Ant tending did not affect the body mass of adult males or the developmental time of either male or female larvae. Thus, tending by C. japonicus could increase the fitness of P. argyrognomon praeterinsularis by increasing the mass of females without prolonging the duration of vulnerable immature stages, because larger females generally lay more eggs. This means that even facultatively myrmecophilous lycaenids might gain fitness benefits from particular ant species, which could be important in the conservation and management of at-risk species of facultatively myrmecophilous lycaenids.