Co-phylogeny of a hyper-symbiotic system: Endosymbiotic bacteria (Gammaproteobacteria), chewing lice (Insecta: Phthiraptera) and birds (Passeriformes).

Chewing lice are hosts to endosymbiotic bacteria as well as themselves being permanent parasites. This offers a unique opportunity to examine the cophylogenetic relationships between three ecologically interconnected organismal groups: birds, chewing lice, and bacteria. Here, we examine the cophylogenetic relationships between lice in the genus Guimaraesiella Eichler, 1949, their endosymbiotic Sodalis-allied bacteria, and a range of bird species from across South China. Both event and distance-based cophylogenetic analyses were explored to compare phylogenies of the three organismal groups. Pair-wise comparisons between lice-endosymbionts and bird-endosymbionts indicated that their evolutionary histories are not independent. However, comparisons between lice and birds, showed mixed results; the distance-based method of ParaFit indicated that their evolutionary histories are not independent, while the event-based method of Jane indicated that their phylogenies were no more congruent than expected by chance. Notably, louse host-switching does not seem to have affected bacterial strains, as conspecific lice sampled from distantly related hosts share bacteria belonging to the same clade.

Lice (Phthiraptera, Amblycera, Ischnocera) collected on the birds in the Aras basin in Igdir Province, Türkiye with new records and new host associations.

Chewing lice (Phthiraptera, Ischnocera and Amblycera) are permanent ectoparasites of birds and primarly feed on the feathers and scales of birds. To detect the chewing lice species found on birds in Aras basin, Igdir, Türkiye, a total of 240 birds represented by 61 species belonging to 30 families in 13 orders were examined during the 2021 bird migration season. A total of 531 (186 females, 136 males and 209 nymphs) lice were collected from 75 individuals (31,25% of birds examined) of 26 species, 21 families and 10 orders. Thirty-one lice species (11 amblyceran and 20 ischnoceran species) in 22 genera were identified. Of these, 15 lice species were reported for the first time in Türkiye, namely Cuculiphilus fasciatus, Pseudomenopon qadrii, Philopterus sp., Ricinus serratus, Philopterus picae, Rostrinirmus buresi, Sturnidoecus sp., Philopterus excisus, Philopterus microsomaticus, Philopterus coarctatus, Brueelia fuscopleura, Sturnidoecus pastoris, Brueelia currucae, Penenirmus auritus and Strigiphilus tuleskovi. In addition, new host associations were reported for the lice species Kurodaia fulvofasciata, Degeeriella rufa and Myrsidea rustica.

D-limonene nanoemulsion: lousicidal activity, stability, and effect on the cuticle of Columbicola columbae.

The current study was conducted to investigate the efficacy and stability of D-limonene (DL) and its nanoemulsion (DLN) against pigeon feather lice (Columbicola columbae) and their mode of action. DL pure form and DLN were prepared and characterized freshly and after storage for 50 days. In vitro bioassay on live lice was conducted with different concentrations of DL, DLN, and deltamethrin (DM). The results revealed significant mortality rates in the DL-, DLN-, DM-treated groups when compared with the control (p < 0.05). The scanning electron micrographs of lice treated with DL and DLN revealed collapsed bodies with destruction in the cuticle of the mouthparts and damaged antennae. The 50 days stored DLN showed stability in their effectiveness when compared with the freshly prepared formulation. DL and DLN caused significant inhibition (p ≤ 0.05) in acetylcholinesterase activity (AchE). Malondialdehyde level (MDA) was significantly increased while glutathione was significantly decreased in DL- and DLN-treated lice. In conclusion, DL and DLN have significant lousicidal activities. DLN showed better stability than DL after storage for 50 days. In addition, the mode of action of DL may associate with its effect on the cuticle of the lice body, inhibition of AchE, and increasing oxidative stress in the treated lice.

Chewing lice of passerine birds in reed beds in Slovakia, with a special focus on Panurus biarmicus.

A total of 1185 passerine birds representing five species were examined for chewing lice in reed beds in southwestern Slovakia in spring (April) 2008, 2009 and 2016. Additional collecting focused only on chewing lice from Panurus biarmicus (Linnaeus, 1758) (Passeriformes: Panuridae) was carried out in spring (April), summer (July) and autumn (October) 2019. A total of 283 (24%) birds were parasitized by 10 species of chewing lice of four genera: Penenirmus, Menacanthus, Philopterus, and Brueelia. Most birds showed only very light (1-10 lice/host; 74%) to light infestations (11-20 lice/host; 16%). The authors found significantly higher prevalences and mean abundances of chewing lice on residents/short-distance migrants, that is, P. biarmicus, Acrocephalus melanopogon (Temminck, 1823) (Passeriformes: Acrocephalidae), than on long-distance migratory birds, that is, Acrocephalus scirpaceus (Hermann, 1804), Acrocephalus schoenobaenus (Linnaeus, 1758) (Passeriformes: Acrocephalidae), Locustella luscinioides (Savi, 1824) (Passeriformes: Locustellidae). No significant difference was found in the total mean intensity of chewing lice between these two groups of birds. Ischnoceran lice were more prevalent and abundant than amblyceran lice on residents and short-distance migrants, whereas the opposite was found on bird species that migrate long distances. A total of 146 (58%, n = 251) P. biarmicus were parasitized by 1490 chewing lice. Males of P. biarmicus showed higher prevalence and mean abundance than females with gradually descending values of prevalence, mean abundance and mean intensity from spring to autumn. The knowledge of the occurrence and population dynamics of lice on wild passerine birds can be useful in endangered species conservation programs and can also be applied to captive passerine birds, which may be analogous to resident birds in this sense.

Parasitological survey of coastal birds from the Magellanic coast, Southwestern Atlantic Ocean.

Ecto and endoparasites of four species of coastal birds, Haematopus ater, Larus dominicanus, Leucophaeus scoresbii (Charadriiformes), and Lophonetta specularioides (Anseriformes), are reported from Puerto Deseado on the Patagonian coast, Argentina. Only H. ater was infested with lice (Phthiraptera), belonging to 2 species (Ischnocera, Amblycera). A total of 19 helminth species were found parasitizing the coastal birds studied: 4 cestodes (1 Tetrabothriidae, 3 Cyclophyllidea); 11 trematodes (2 Gymnophallidae, 3 Microphallidae, 2 Notocotylidae, 1 Philophthalmidae, 2 Renicolidae, 1 Schistosomatidae); 3 nematodes (1 Anisakidae, 2 Acuariidae); and 1 acanthocephalan (Polymorphidae). Although some isolated records have been previously reported for these birds, the present work provides a parasitofauna study for H. ater, L. scoresbii, and L. specularioides for the first time. Endoparasites reflected the feeding habits of the birds; the parasite assemblage of L. dominicanus was the richest, indicating their wide prey spectrum and the diversity of the habitats frequented. A great species richness of trematodes, whose life cycles are partially known, suggests that L. specularioides feeds upon crustaceans and small bivalves. The blackish oystercatcher H. ater preys upon the limpet Nacella magellanica which hosts two larval trematodes corresponding to the adults found parasitizing it.

Molecular phylogenetics of the avian feather louse Philopterus-complex (Phthiraptera: Philopteridae).

The avian feather louse Philopterus-complex (Phthiraptera: Ischnocera: Philopteridae) currently contains 12 genera that have been grouped together because of shared morphological characteristics. Although previously lumped into a single genus (Philopterus), more recent morphological treatments have separated the group into several different genera. Here we evaluate the status of these genera using DNA sequence data from 118 ingroup specimens belonging to ten genera in the Philopterus-complex: Australophilopterus Mey, 2004, Cinclosomicola Mey 2004, Clayiella Eichler, 1940, Corcorides Mey, 2004, Mayriphilopterus Mey, 2004, Paraphilopterus Mey 2004, Philopteroides Mey 2004, Philopterus Nitzsch, 1818, Tyranniphilopterus Mey, 2004, and Vinceopterus Gustafsson, Lei, Chu, Zou, and Bush, 2019. Our sampling includes 97 new louse-host association records. Our analyses suggest that the genus Debeauxoecus Conci, 1941, parasitic on pittas (Aves: Pittidae), is outside of the Philopterus-complex, and that there is strong support for the monophyly of a group containing the remaining genera from the complex. Some diverse genera, such as Philopterus (sensu stricto) and Mayriphilopterus are supported as monophyletic, whereas the genera Australophilopterus, Philopteroides, and Tyranniphilopterus are not. The present study is the largest phylogenetic reconstruction of avian lice belonging to the Philopterus-complex to date and suggests that further generic revision is needed in the group to integrate molecular and morphological information.

Pichia bovicola sp. nov., a yeast species associated with small-intestinal content of cattle.

Three yeast strains, DMKU-MP6-4T, DMKU-MP2-6 and DMKU-MP5-1, were isolated from the small-intestinal content or Pia of cattle in Thailand during the investigation of yeast diversity in this habitat. According to the D1/D2 domain of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) region sequence analysis, these strains represent a novel yeast species in the genus Pichia. The species produced one to four ascospores per ascus with spherical to ellipsoidal shape and heterogenous in terms of size. These three strains were identical and differed from their closely related species, Pichia exigua NRRL Y-10920T by 2% (six nucleotide substitutions and five gaps) in the D1/D2 domain of the large subunit rRNA gene, while ITS region sequences differed by 3.1% (16 nucleotide substitutions and 27 gaps), 3.7% (19 nucleotide substitutions and 28 gaps) and 3.1% (16 nucleotide substitutions and 27 gaps) for DMKU-MP6-4T, DMKU-MP2-6 and DMKU-MP5-1, respectively. The name, Pichia bovicola, is proposed to accommodate these species. The holotype is DMKU-MP6-4T (TBRC 15616T=PYCC 8905T).

Development of molecular detection methods of Bovicola ovis from sheep fleece.

The sheep body louse (Bovicola ovis) commonly referred to as sheep lice are small chewing ectoparasites of sheep. Infection results in significant economic costs to the Australian sheep industry due to reduced wool quality caused by chronic itching from sheep rubbing and biting fleece. Treatment relies on use of insecticides; however, resistance has developed against pyrethroid and other insect growth regulator lousicides. There is urgent need to develop cost-effective lice management to reduce the use of insecticides, with the application of insecticidal treatments only applied when an infestation is detected. However, the current detection method relies on fleece parting for detection of B. ovis which is highly dependent on the skill of the inspector, the number of sheep examined, and the prevalence and severity of the infestation. To improve B. ovis detection, a highly sensitive (5 × 10-8 ng/µL) and specific multiplex quantitative PCR which simultaneously detects sheep lice and sheep DNA was developed. In addition, a B. ovis loop-mediated isothermal amplification (LAMP) assay was developed for field use. The B. ovis LAMP (Bov-LAMP) assay was optimized to reliably detect B. ovis from wool samples down to 5 × 10-6 ng/µL, with time to positive (Tp) < 10 min. Both assays demonstrate high sensitivity and specificity, enabling rapid identification of B. ovis DNA from sheep fleece samples and have the capacity to be used for ongoing management and surveillance of B. ovis in Australian sheep flocks.

Integrative taxonomic description of the chewing louse Tricholipeurus lipeuroides infesting Odocoileus virginianus veraecrucis white-tailed deer in Veracruz, Mexico.

Chewing lice (Phthiraptera: Amblycera, Ischnocera) represent a component of the ectoparasite fauna associated with large sized mammals as deers. However, the diversity of chewing louse species infesting deer remains to be fully characterized in the Neotropics. Little is known about the chewing lice infesting the extant fourteen subspecies of white-tailed deer (Odocoileus virginianus) in Mexico. Known to infest white-tailed deer (WTD) in Canada and the United States (U.S.), Tricholipeurus lipeuroides is a chewing louse species that was originally described in the nineteenth century infesting O. v. mexicanus in Mexico. For the first time, infestation of O. v. veraecrucis, a Neotropical WTD subspecies in Mexico, with T. lipeuroides is reported herein. An integrative taxonomic approach was taken by combining morphological and molecular analyses to describe the T. lipeuroides infestion of O. v. veraecrucis. Ecological parameters of the T. lipeuroides infestations were also calculated. The prevalence was 91.7% of the 56 O. v. veraecrucis (29 females and 27 males) inspected while under chemical restraint that were sampled at 3 sites in the central region of Veracruz state in Mexico. The amplification and sequencing of previously reported T. lipeuroides Cytochrome Oxidase Subunit I gene confirmed the identity of all the chewing louse life stages. These results are discussed in the context of comparative analyses on the emergence of novel chewing lice-deer associations.

Sex-dependent changes in the louse abundance of red-footed falcons (Falco vespertinus).

Permanent ectoparasites live in stable environments; thus, their population dynamics are mostly adapted to changes in the host life cycle. We aimed to investigate how static and dynamic traits of red-footed falcons interplay with the dynamics of their louse subpopulations during breeding and how they affect the colonisation of new hosts by lice. We sampled red-footed falcon (Falco vespertinus) nestlings (two breeding seasons) and adults (one breeding season) in southern Hungary. The mean abundance of Colpocephalum subzerafae and Degeeriella rufa lice on the nestlings was modelled with generalized linear mixed models using clutch size and host sex in interaction with wing length. For adults, we used wing length and the number of days after laying the first egg, both in interaction with sex. D. rufa abundances increased with the nestlings' wing length. In one year, this trend was steeper on females. In adult birds, both louse species exhibited higher abundances on females at the beginning, but it decreased subsequently through the breeding season. Contrarily, abundances were constantly low on adult males. Apparently, D. rufa postpones transmission until nestlings develop juvenile plumage and choose the more feathered individual among siblings. The sexual difference in the observed abundance could either be caused by the different plumage, or by the females' preference for less parasitized males. Moreover, females likely have more time to preen during the incubation period, lowering their louse burdens. Thus, sex-biased infestation levels likely arise due to parasite preferences in the nestlings and host behavioural processes in the adult falcons.

Rapid detection of Bovicola ovis using colourimetric loop-mediated isothermal amplification (LAMP): a potential tool for the detection of sheep lice infestation on farm.

The sheep body louse, Bovicola ovis (B. ovis), is one of the most significant ectoparasites affecting Australia's sheep flocks. Despite this, detection methods for B. ovis infestation are limited to visual inspection and ELISA. A colourimetric loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of B. ovis DNA. Diagnostic sensitivity and specificity of LAMP were compared with those of visual inspection and PCR and validated using field samples collected from 22 farms. Two different DNA extraction methods using a commercial kit and a boiling method were also compared. The highest sensitivity and specificity were observed when PCR was used and DNA was extracted using a commercial kit. Compared with PCR, the LAMP assay demonstrated a sensitivity and specificity of 90% and 92% when DNA was extracted by a commercial kit and 100% and 75% when DNA was extracted by the boiling method, respectively. The LAMP test developed in this study could potentially serve as a point-of-care diagnostic tool for monitoring of sheep flocks as well as surveillance of B. ovis populations.

Loss of genetic diversity, recovery and allele surfing in a colonizing parasite, Geomydoecus aurei.

Understanding the genetic consequences of changes in species distributions has wide-ranging implications for predicting future outcomes of climate change, for protecting threatened or endangered populations and for understanding the history that has led to current genetic patterns within species. Herein, we examine the genetic consequences of range expansion over a 25-year period in a parasite (Geomydoecus aurei) that is in the process of expanding its geographic range via invasion of a novel host. By sampling the genetics of 1,935 G. aurei lice taken from 64 host individuals collected over this time period using 12 microsatellite markers, we test hypotheses concerning linear spatial expansion, genetic recovery time and allele surfing. We find evidence of decreasing allelic richness (AR) with increasing distance from the source population, supporting a linear, stepping stone model of spatial expansion that emphasizes the effects of repeated bottleneck events during colonization. We provide evidence of post-bottleneck genetic recovery, with average AR of infrapopulations increasing about 30% over the 225-generation span of time observed directly in this study. Our estimates of recovery rate suggest, however, that recovery has plateaued and that this population may not reach genetic diversity levels of the source population without further immigration from the source population. Finally, we employ a grid-based sampling scheme in the region of ongoing population expansion and provide empirical evidence for the power of allele surfing to impart genetic structure on a population, even under conditions of selective neutrality and in a place that lacks strong barriers to gene flow.

Chewing lice from high-altitude and migrating birds in Yunnan, China, with descriptions of two new species of Guimaraesiella.

In total, 366 birds representing 55 species in 24 families and eight orders, were examined for chewing lice (Phthiraptera: Amblycera, Ischnocera) in two high-altitude localities in Yunnan Province, China. In Ailaoshan, almost all of the birds examined were resident passeriforms, of which 36% were parasitized by chewing lice. In Jinshanyakou, most birds were on migration, and included both passerine and non-passerine birds. Of the passerine birds caught in Jinshanyakou, only one bird (0.7%) was parasitized by chewing lice. The prevalence of Myrsidea and Brueelia-complex lice on birds caught in Ailaoshan was higher than in previous reports. Of the chewing lice identifiable to species level, three represent new records for China: Actornithophilus hoplopteri (Mjöberg, 1910), Maculinirmus ljosalfar Gustafsson & Bush, 2017 and Quadraceps sinensis Timmermann, 1954. In total, 17 new host records are included, of which we describe two as new species in the Brueelia-complex: Guimaraesiella (Cicchinella) ailaoshanensis sp. nov. ex Schoeniparus dubius dubius (Hume, 1874) and G. (C.) montisodalis sp. nov. ex Fulvetta manipurensis tonkinensis Delacour & Jabouille, 1930. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:9FC3D8EE-2CED-4DBE-A1DB-471B71260D27.

Seven new species of Resartor Gustafsson et Bush, 2017 (Phthiraptera: Ischnocera: Philopteridae) from Asian 'babblers' (Passeriformes: Leiothrichidae, Paradoxornithidae).

Seven new species of chewing lice in the genus Resartor Gustafsson et Bush, 2017 are described and illustrated. They are: Resartor albofulvus sp. n. ex Heterophasia desgodinsi desgodinsi (Oustalet); Resartor apimimus sp. n. ex Heterophasia picaoides wrayi (Ogilvie-Grant); Resartor aterrimus sp. n. ex Minla ignotincta mariae La Touche; Resartor extraneus sp. n. ex Lioparus chrysotis swinhoii (Verreaux); Resartor guangxiensis sp. n. ex Trochalopteron milnei sinianum Stresemann; Resartor longisuturalis sp. n. ex Actinodura cyanouroptera wingatei (Ogilvie-Grant); Resartor seminudus sp. n. ex Leiothrix argentauris tahanensis (Yen). All species differ in the shape of the head, shape of the male genitalia and abdominal chaetotaxy. A checklist and a key to the species of Resartor are provided.

Residual and ovicidal efficacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus.

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N-lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.

New host/parasite record for very rare chewing louse Cuculotogaster arabicus (Clay, 1938) (Phthiraptera: Ischnocera) on endemic mountain partridges of Arabian Peninsula.

The endemic avifauna of Arabia is unique and characteristic through the whole region. Little is known about these birds and their ectoparasites. The Arabian partridge Alectoris melanocephala (Rüppell, 1835) and Philby's partridge Alectoris philbyi Lowe, 1934 are two endemic species which are distributed through the Sarawat Mountains in Saudi Arabia and Yemen. Captive breeding population of these birds were examined for chewing lice at the National Wildlife Research Center (NWRC) near Ta'if. Only one rare species of louse, Cuculotogaster arabicus (Clay 1938), was found to infest these birds. The occurrence of such species is considered a first record of the genus Cuculotogaster from Saudi Arabia, and the association of C. arabicus with Philby's partridge is considered a new host/parasite association. The clear diagnostic characters, high definition photos, and drawing of male genitalia are available through this paper.

The Chewing Lice (Insecta, Phthiraptera) Fauna of the Swainson's Warbler, Limnothlypis swainsonii (Aves, Parulidae).

We examined Swainson's warblers (Limnothlypis swainsonii (Audubon, 1834), Aves: Parulidae) for lice fauna during 2 yr at three study sites in Arkansas, USA. A total of 66 individuals were examined; eight birds (10.6%) were parasitized with 16 lice of two new species belonging to two genera Myrsidea Waterson, 1915 (Amblycera: Menoponidae) and Brueelia Kéler, 1936 (Ischnocera: Philopteridae). Parasitological parameter data are given on the prevalence of lice on Swainson's warblers. Species descriptions and illustrations are provided for Myrsidea bensoni sp. nov. and Brueelia limnothlypiae sp. nov.; including a key for females of the genus Myrsidea that parasitize Parulidae (Passeriformes).

New records of species of Saemundssonia (Insecta: Phthiraptera: Philopteridae) infesting breeding terns in the Arabian Peninsula, with notes on their phylogeny and ecology.

Six species of terns, which breed on the Arabian Peninsula, were examined for head chewing lice of the genus Saemundssonia in four different islands around the coasts of Saudi Arabia, both in the Red Sea and in the Arabian Gulf. Four louse species were collected: Saemundssonia laticaudata, Saemundssonia melanocephalus, Saemundssonia meridiana and Saemundssonia sternae, of which three are recorded for the first time from this region. Also, we record three new host-louse associations for the world-Saemundssonia laticaudata and Saemundssonia sternae from white-cheeked terns and Saemundssonia melanocephalus from Saunders's terns-including a host-switch event of Saemundssonia laticaudata on white-cheeked terns in the Karan Island population. Gene bank data for the COI gene from seven species of Saemundssonia that infest marine birds were used to propose evolutionary trees using two different statistical methods: maximum parsimony (MP) and neighbour joining (NJ). The result indicated that the tree which was produced by NJ is likely to be more accurate as it appeared more compatible with hosts' phylogeny. The trees indicate relationships between tern Saemundssonia and congeneric species from other marine birds, especially from gulls. An ANOVA was also conducted to test the mean parasite load for each tern species studied, and results indicate that there is a relation between louse loads and colonization behaviour of the hosts. Data from lice examined and illustrations of lice and their hosts are also included.

Mechanisms underlying parasite infection: influence of host body mass and age on chewing louse distribution among brown-headed cowbirds.

Animal populations exhibit various patterns in ectoparasite distribution across different sexes and age classes, and numerous factors can potentially contribute to ectoparasite abundance and distribution. We examined the influence of host body size and age on the distribution of a chewing louse among brown-headed cowbirds. Differences in louse prevalence (males 62.9 ± 2.8%, females 47.5 ± 4.9%) and intensity (males 15.42 ± 1.51, females 9.04 ± 1.69) were primarily driven by differences in host body mass and not host sex. Larger birds had larger louse infra-populations, which likely translated into a lower risk of local extinction, a possible explanation for higher louse prevalence observed among larger birds. Among males, younger individuals showed higher louse prevalence (70.21 ± 4.72%) compared to older males (59.36 ± 3.59). We speculate that this pattern is likely driven by behavioural difference and not body size, with young males spending relatively more time foraging in large groups, increasing their risk of louse transmission. By examining the mechanisms that underlie the sex- and age-biased infections observed in natural populations, we can better identify the hosts most responsible for parasite transmission.

Unprecedented conformational flexibility revealed in the ligand-binding domains of the Bovicola ovis ecdysone receptor (EcR) and ultraspiracle (USP) subunits.

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Šresolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.