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Alternative life-history strategies (ALHS) are genetic polymorphisms generating phenotypes differing in life histories that generally arise due to metabolic resource allocation tradeoffs. Althouigh ALHS are often be limited to a single sex or populations of a species, they can, in rare cases, be found among several species across a genus. In the butterfly genus Colias, at least a third of the species have a female limited ALHS called Alba. While many females develop brightly pigmented wings, Alba females reallocate nitrogen resources used in pigment synthesis to reproductive development, producing white-winged, more fecund females. Whether this ALHS evolved once or many times, and whether it has moved among species via introgression or been maintained via long-term balancing selection, has not been established. Answering these questions presents an opportunity to investigate the genetic basis and evolutionary forces acting upon ALHS, which have rarely been studied at a genus level. Here we identify the genetic locus of Alba in a second Colias species, allowing us to compare this with previous results in a larger phylogenetic context. Our findings suggest Alba has a singular origin and has been maintained in Colias through a combination of balancing selection and introgression for nearly one million years and at least as many generations. Finally, using CRISPR/Cas9 deletions in the cis-regulatory region of the Alba allele, we demonstrate that the Alba allele is a modular enhancer for the BarH1 gene and is necessary for the induction of the ALHS, which potentially facilitates its long-term persistence in the genus.

BACKGROUND: MenB-FHbp is a licensed meningococcal B vaccine targeting factor H-binding protein. Two phase 3 studies assessed the safety of the vaccine and its immunogenicity against diverse strains of group B meningococcus.METHODS: We randomly assigned 3596 adolescents (10 to 18 years of age) to receive MenB-FHbp or hepatitis A virus vaccine and saline and assigned 3304 young adults (18 to 25 years of age) to receive MenB-FHbp or saline at baseline, 2 months, and 6 months. Immunogenicity was assessed in serum bactericidal assays that included human complement (hSBAs). We used 14 meningococcal B test strains that expressed vaccine-heterologous factor H-binding proteins representative of meningococcal B epidemiologic diversity; an hSBA titer of at least 1:4 is the accepted correlate of protection. The five primary end points were the proportion of participants who had an increase in their hSBA titer for each of 4 primary strains by a factor of 4 or more and the proportion of those who had an hSBA titer at least as high as the lower limit of quantitation (1:8 or 1:16) for all 4 strains combined after dose 3. We also assessed the hSBA responses to the primary strains after dose 2; hSBA responses to the 10 additional strains after doses 2 and 3 were assessed in a subgroup of participants only. Safety was assessed in participants who received at least one dose.RESULTS: In the modified intention-to-treat population, the percentage of adolescents who had an increase in the hSBA titer by a factor of 4 or more against each primary strain ranged from 56.0 to 85.3% after dose 2 and from 78.8 to 90.2% after dose 3; the percentages of young adults ranged from 54.6 to 85.6% and 78.9 to 89.7%, after doses 2 and 3, respectively. Composite responses after doses 2 and 3 in adolescents were 53.7% and 82.7%, respectively, and those in young adults were 63.3% and 84.5%, respectively. Responses to the 4 primary strains were predictive of responses to the 10 additional strains. Most of those who received MenB-FHbp reported mild or moderate pain at the vaccination site.CONCLUSIONS: MenB-FHbp elicited bactericidal responses against diverse meningococcal B strains after doses 2 and 3 and was associated with more reactions at the injection site than the hepatitis A virus vaccine and saline. (Funded by Pfizer; ClinicalTrials.gov numbers, NCT01830855 and NCT01352845 ).

Leptoglossus occidentalis causes significant damage in conifer seed orchards. Host selection by L. occidentalis is not completely understood. Earlier research has demonstrated a preference for certain clones of Pinus contorta, indicating that L. occidentalis responds to chemical or physical cues. The present study aimed to test whether L. occidentalis shows clonal preference across years, and to examine whether the host cues responsible for this could be identified. Surveys were conducted in a lodgepole pine seed orchard in British Columbia in 2008 and 2009. Clones were ranked based on the proportion of their ramets on which L. occidentalis was observed. Ramets were divided into three classes: (i) preferred clones with seed bugs; (ii) preferred clones without seed bugs; and (iii) nonpreferred clones with zero or very low numbers of seed bugs. From each clone, we measured infrared radiation emitted from cones, cone monoterpenes, cone size and numbers of cones per tree. Clone preference was consistent between 2008 and 2009. Clone preference classes differed significantly in α-pinene and δ-3 carene and limonene. Leptoglossus occidentalis was found more frequently on clones with cones of greater diameter and weight. Infrared radiation did not differ between clone preference classes, indicating that it is not used in host acceptance.

International audience; Retracing the routes of invasions and determining the origins of invading species is often critical in understanding biological invasions. The Western conifer seed bug, Leptoglossus occidentalis, an insect native of western North America, was first accidentally introduced to eastern North America and then to Europe. The colonization of the entire European continent occurred in ca. 10–15 years, probably promoted by independent introductions in different parts of Europe. A multi-marker approach (mtDNA and microsatellites) combined with approximate Bayesian computation analyses was used to track the origin of European populations and to determine whether this rapid invasion was caused by multiple introductions. Our results show that at least two independent introductions of L. occidentalis have occurred in Europe. Moreover, the analyses showed a stronger genetic similarity of European invasive populations with the eastern North American populations than with those of the native range, suggesting that invasive North American population acted as a bridgehead for European invasion. The results also revealed that natural dispersal as well as human-mediated transportations as hitchhikers probably enhanced the rapid spread of this invasive pest across Europe. This study illustrates the complexity of a rapid invasion and confirms that bridgehead and multiple introductions have serious implications for the success of invasion.

Leptoglossus occidentalis Heidemann (Heteroptera, Coreidae) is a harmful insect to cones and seeds of several conifers in North America, and was accidentally introduced into Europe where it rapidly spread to many countries. In Central Italy, the pest damaged cones and seeds of Pinus pinea L., leading to a severe reduction of pine nut production. Gryon pennsylvanicum (Ashmead) (Hymenoptera, Platygastridae) is the most common egg-parasitoid of L. occidentalis in British Columbia. This natural enemy has been successfully reared under laboratory conditions. However, the possibility to arrange a sufficient large number of specimens is crucial for the success in biological control. In the present work, stored L. occidentalis eggs at fridge and freezing temperatures of +4, −20 °C, and ultra-low temperatures of −80 and −140 °C for different times up to one year, were tested for parasitization success. Results showed that G. pensylvanicum can exploit successfully host eggs stored for long time when stored at ultra-low temperatures and that storage did not affect fitness of the first and second generation. For shorter periods of time, the use of +4 °C for store eggs did not vary significantly from eggs stored at ultra-low temperatures.

Wildlife diseases have been implicated in the declines and extinctions of several species. The ability of a pathogen to persist outside its host, existing as an ''environmental reservoir'', can exacerbate the impact of a disease and increase the likelihood of host extinction. Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats, has been found in cave soil during the summer when hibernating bats had likely been absent for several months. However, whether the pathogen can persist over multiple years in the absence of bats is unknown, and long-term persistence of the pathogen can influence whether hibernacula where bats have been locally extirpated due to disease can be subsequently recolonized. Here, we show that P. destructans is capable of long-term persistence in the laboratory in the absence of bats. We cultured P. destructans from dried agar plates that had been kept at 5C and low humidity conditions (30-40% RH) for more than 5 years. This suggests that P. destructans can persist in the absence of bats for long periods which may prevent the recolonization of hibernation, sites where bat populations were extirpated. This increases the extinction risk of bats affected by this disease.

The molecular tools of genomics have great power to reveal patterns of genetic difference within or among species, but must be complemented by the mechanistic study of the genetic variants found if these variants' evolutionary meaning is to be well understood. Central to this purpose is knowledge of the organisms' genotype-phenotype-environment interactions, which embody biological adaptation and constraint and thus drive natural selection. The history of this approach is briefly reviewed. Strategies embracing the complementarity of genomics and specific-gene studies in evolution are considered. Implementation of these strategies, and examples showing their feasibility and power, are discussed. Initial generalizations emphasize: (1) reproducibility of adaptive mechanisms; (2) evolutionary co-importance of variation in protein sequences and expression; (3) refinement of rudimentary molecular functions as an origin of evolutionary innovations; (4) identification of specific-gene mechanisms as underpinnings of genomic or quantitative genetic variation; and (5) multiple forms of adaptive or constraining epistasis among genes. Progress along these lines will advance understanding of evolution and support its use in addressing urgent medical and environmental applications.

Leptoglossus occidentalis Heidemann (Heteroptera, Coreidae) is native to Western North America and is a serious pest for seed production of conifers. The pest was accidentally introduced into Europe in the 1990s. Since then, seed loss has been recorded in Pinus pinea (L.) forests, with a negative impact on the commercial production of pine nuts. Classical biological control of this pest in P. pinea stands is an attractive proposition. Previous work showed that the egg-parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera, Platygastridae) had promising life history traits in laboratory studies using L. occidentalis eggs as host. In the present work, the effect of host egg age on parasitization rate was evaluated in the laboratory, using choice and no-choice tests. Host eggs ranged in age from < 24 h to within a day of hatching. Results showed that parasitization rate, juvenile survival rate, sex ratio, and longevity of female G. pennsylvanicum were not significantly affected by the age of the host eggs. However, egg-parasitoid development time was longer in older host eggs, and females were smaller than those that developed in younger host eggs. Parasitization behaviors (drumming, oviposition, and marking) were not affected by the age of the host. G. pennsylvanicum females tended to parasitize all available host eggs within a cluster before moving to a new cluster, without displaying a preferences for host egg age. The ability to exploit host eggs of any age class improves the prospect of successful classical biological control using this egg-parasitoid.

Ernst Mayr proposed a distinction between “proximate”, mechanistic, and “ultimate”, evolutionary, causes of biological phenomena. This dichotomy has influenced the thinking of many biologists, but it is increasingly perceived as impeding modern studies of evolutionary processes, including study of “niche construction” in which organisms alter their environments in ways supportive of their evolutionary success. Some still find value for this dichotomy in its separation of answers to “how?” versus “why?”questions about evolution. But “why is A?” questions about evolution necessarily take the form “how does A occur?”, so this separation is illusory. Moreover, the dichotomy distorts our view of evolutionary causality, in that, contra Mayr, the action of natural selection, driven by genotype-phenotype-environment interactions which constitute adaptations, is no less “proximate” than the biological mechanisms which are altered by naturally selected genetic variants. Mayr’s dichotomy thus needs replacement by more realistic, mechanistic views of evolution. From a mechanistic viewpoint, there is a continuum of adaptations from those evolving as responses to unchanging environmental pressures to those evolving as the capacity for niche construction, and intermediate stages of this can be identified. Some biologists postulate an association of “phenotypic plasticity” (phenotype-environment covariation with genotype held constant) with capacity for niche construction. Both “plasticity” and niche construction comprise wide ranges of adaptive mechanisms, often fully heritable and resulting from case-specific evolution. Association of “plasticity” with niche construction is most likely to arise in systems wherein capacity for complex learning and behavioral flexibility have already evolved.

The parasitoid complex of the western conifer seed bug, Leptoglossus occidentalis, was studied in British Columbia and California on Pinus monticola and P. contorta var. latifolia. Three egg-parasitoid species were identified: Gryon pennsylvanicum (Hymenoptera: Platygastridae), Ooencyrtus johnsoni (Hymenoptera: Encyrtidae) and Anastatus pearsalli (Hymenoptera: Eupelmidae). Leptoglossus occidentalis represents a new host record for O. johnsoni, and California is a new distribution record for G. pennsylvanicum. Gryon pennsylvanicum was the dominant species during the entire oviposition period of the bug, reaching peak parasitization levels in July (25%). Ooencyrtus johnsoni and A. pearsalli emerged only from eggs collected on Pinus monticola. The parasitization level by G. pennsylvanicum recorded on the two different Pinaceae hosts did not show any statistically significant difference. No tachinid eggs or exit holes by tachinid larvae were observed in any adult L. occidentalis collected in either British Columbia or California. The data collected encouraged the introduction of G. pennsylvanicum in Italy where L. occidentalis has become a serious pest. Laboratory host range assessment with this Platygastridae, in preparation for its field release in Italy, has already started.

Leptoglossus occidentalis Heidemann (Heteroptera, Coreidae), the western conifer seed bug, is a serious conifer seed pest in North America; after its accidental introduction in Italy, it has spread to several European countries. In Central Italy, severe seed loss was recorded in stone pine forests (Pinus pinea). Classical biological control against L. occidentalis was evaluated by performing laboratory tests on Gryon pennsylvanicum (Ashmead), the most important hymenopteran egg parasitoid of the pest in North America, to acquire biological and demographical parameters. Tests were performed in climatic chambers using glass tubes to house specimens at 26 ± 1 °C, 75 ± 5% RH and 16:8 L:D and supplying honey drops as food for adults. G. pennsylvanicum reared on eggs of L. occidentalis has a high fecundity rate (144.55 eggs/female) and 90% of the total number of eggs are laid in the first two weeks of oviposition. Mean oviposition period is 23 days. Parasitoid females live a mean of 40 days, and longevity is higher in host deprived females. The intrinsic rate of increase (rm) was calculated to be 0.206. Mean sex ratio throughout a female’s life span was 70.42% females, though at the start of oviposition it was over 87% females. Adults survived only four days without honey as food, but up to 50 days with food; water did not influence longevity. Females lived significantly longer than males. Fecundity was negatively affected by duration of host deprivation, nevertheless, host-deprived females retained the ability to parasitize host eggs, and without a substantial sex ratio change in offspring. On the whole, G. pennsylvanicum shows potential for use in classical biological control against L. occidentalis. Several factors contribute to its suitability for biological control like high reproductive rate, high sex ratio, long-lived adults, and the ability to maintain reproductive potential during periods of host deprivation with a no substantial change in the sex ratio.

Little is known of intron sequences' variation in cases where eukaryotic gene coding regions undergo strong balancing selection. Phosphoglucose isomerase, PGI, of Colias butterflies offers such a case. Its 11 introns include many point mutations, insertions, and deletions. This variation changes with intron position and length, and may leave little evidence of homology within introns except for their first and last few basepairs. Intron position is conserved between PGIs of Colias and the silkmoth, but no intron sequence homology remains. % GC content and length are functional properties of introns which can affect whole-gene transcription; we find a relationship between these properties which may indicate selection on transcription speed. Intragenic recombination is active in these introns, as in coding sequences. The small extent of linkage disequilibrium (LD) in the introns decays over a few hundred basepairs. Subsequences of Colias introns match subsequences of other introns, untranslated regions of cDNAs, and insect-related transposons and pathogens, showing that a diverse pool of sequence fragments is the source of intron contents via turnover due to deletion, recombination, and transposition. Like Colias PGI's coding sequences, the introns evolve reticulately with little phylogenetic signal. Exceptions are coding-region allele clades defined by multiple amino acid variants in strong LD, whose introns are closely related but less so than their exons. Similarity of GC content between introns and flanking exons, lack of small introns despite mutational bias toward deletion, and findings already mentioned suggest constraining selection on introns, possibly balancing transcription performance against advantages of higher recombination rate conferred by intron length.

Recent efforts to catalogue global biodiversity using genetic techniques have uncovered a number of "cryptic" species within morphologically similar populations that had previously been identified as single species. Chlosyne lacinia (Lepidoptera: Nymphalidae), with a range extending from the Southwest U.S. to South America, is one of the most phenotypically variable and broadly distributed butterfly species in the New World. We sampled populations of C. lacinia in two temperate locations (California and Arizona) and one tropical location (El Salvador) to determine if cryptic species were present at this scale (temperate vs. tropical). We examined mtDNA sequence variation in COI, COII, the intervening tRNA (Leucine-2), 16S, 12S and an additional intervening tRNA (Valine), accounting for approximately 20% of the mitochondrial genome (3479 bp). Among all C. lacinia individuals, sequence divergence did not exceed 0.0084 compared to a 0.06 estimated divergence between C. lacinia and congener C. leanira. We also found subclade structure which did not clearly correspond to geography or subspecific designation. Though the mitochondrial phylogeny suggests a complex evolutionary history and biogeography, we demonstrate that one C. lacinia species is distributed throughout North and Central America spanning a diverse set of temperate and tropical habitats.

Leptoglossus occidentalis (Heteroptera, Coreidae), a Nearctic species, was accidentally introduced into Northern Italy in the late 1990s, from where it has spread throughout Europe. The bug causes abortion of immature cones of Pinus pinea L., with economic impact on the pine-nut industry. As part of a pest control research program, the egg parasitoid Gryon pennsylvanicum Ashmead (Hymenoptera, Platygastridae) was collected from British Columbia, Canada, and legally introduced to a quarantine climatic chamber in Florence, Italy. The egg parasitoid will be tested against native nontarget heteropterans, an environmental impact assessment will be conducted, and a mass rearing method will be developed if appropriate. The ultimate goal is to release G. pennsylvanicum into Italian P. pinea forests for classical biological control of L. occidentalis.

Within-plant and between-plants distributions of the Cooley spruce gall adelgid, Adelges cooleyi (Gillette), fundatrices were measured in “interior spruce”, Picea glauca × P. engelmannii (Moench) Voss. & Parry (Pinaceae), seed orchards in central British Columbia to develop a quantitative sampling plan. Adelgids (number of fundatrices per shoot) were abundant at the lower and middle crown heights of the tree crowns but scarce at the upper crown height, and more abundant on the ends and central one-third of branches than on the inner portions. The northern and eastern aspects of trees had slightly but significantly more adelgids than did the southern and western aspects, and terminal shoots had more adelgids than did distolateral shoots. Variance was much greater between than within trees. These data were used to create a sampling plan with a sample unit consisting of a single 1-year-old shoot selected from the ends or central area of main branches in the lower or middle portion of each sample tree. Collecting 104 samples required 69 min, and yielded an estimate of the mean number of fundatrices per shoot with a standard error of 10% of the mean.

Foraging animals use diverse cues to locate resources. Common foraging cues have visual, auditory, olfactory, tactile or gustatory characteristics. Here, we show a foraging herbivore using infrared (IR) radiation from living plants as a host-finding cue. We present data revealing that (i) conifer cones are warmer and emit more near-, mid- and long-range IR radiation than needles, (ii) cone-feeding western conifer seed bugs,Leptoglossus occidentalis(Hemiptera: Coreidae), possess IR receptive organs and orient towards experimental IR cues, and (iii) occlusion of the insects' IR receptors impairs IR perception. The conifers' cost of attracting cone-feeding insects may be offset by occasional mast seeding resulting in cone crops too large to be effectively exploited by herbivores.

We study the phylogenetic relationships among some North American Colias (“sulfur”) butterflies, using mitochondrial gene sequences (ribosomal RNA, cytochrome oxidase I + II) totaling about 20% of the mitochondrial genome. We find that (1) the lowland species complex shows a branching order different from earlier views; (2) several montane and northern taxa may be more distinct than in earlier views; (3) one morphologically conservative Holarctic assemblage, C. hecla , is differentiated at the molecular-genetic level into at least three taxa which occupy distinct positions in the phylogeny and are sisters to diverse other taxa. These conclusions, constituting phylogenetic hypotheses, are supported by parsimony, maximum-likelihood, and Bayesian reconstruction algorithms. They are tested formally, by interior branch tests and paired-site tests, against alternative hypotheses derived from conventional species and subspecies naming combinations. In all cases our hypotheses are supported by these tests and the conventional alternatives are rejected. The “barcoding” subset of cytochrome oxidase I sequence identifies only some of the taxa supported by our full data set. Comparison of genetic divergence values among Colias taxa with those among related Pierid butterflies suggests that species radiations within Colias are comparatively younger. This emerging Colias phylogeny facilitates comparisons of genetic polymorphism and other adaptive mechanisms among taxa, thereby connecting micro- and macro-evolutionary processes.

We tested the hypothesis that the western conifer seed bug, Leptoglossus occidentalis Heidemann, uses a substrate-borne vibratory signal for short-range communication. To record such a signal we used computers equipped with data-acquisition hardware and software, microphones sensitive to sonic and (or) ultrasonic frequencies, membrane-type and piezoelectric speakers capable of emitting sonic and ultrasonic sound, and piezoelectric devices capable of emitting low-level, low-frequency vibrations. By tapping their abdomen on substrate, males produced a wide-band vibratory signal 20 dB (sound pressure level; 0 dB = 20 µPa) above ambient sound, with dominant frequencies of 115 ± 10 and 175 ± 15 Hz and a distinct temporal pattern. There was no evidence for (i) ultrasonic signal components; (ii) signals produced by females or nymphs, or (iii) repeated trains of signal pulses. In two-choice arena experiments, males and females preferred the played-back recording of the male-produced substrate-borne signal over silent controls, whereas nymphs showed no preference for either stimulus. In two-choice dowel experiments with hickory wood or lodgepole pine crossbeams, females (unlike males or nymphs) preferred played-back recordings of the same signal over controls. In two-choice field experiments, this signal emitted in the air by piezoelectric devices or transferred through a wire to lodgepole pine branches attracted more L. occidentalis than did silent controls. Our data support the hypothesis that L. occidentalis uses a substrate-borne vibratory signal for short-range communication. The use of such a signal is consistent with reports on communication by other true bug species.

‘Adaptation,’ a central concept in evolutionary biology, represents the degree of suitedness between organisms' characteristics and their environments. It is the cause of differences in organisms' performances of biological tasks that lead throughout their life cycles to reproductive success, the distinct concept of ‘fitness.’ Organisms' characteristics need not be adaptive but may be ‘neutral’ (i.e., they have no consequences for fitness or may be subject to various constraints). Misperceptions of adaptation may result from confusions of terminology. The assumption that all features of organisms are adaptive is common but misleading. Future study of adaptation will include diverse approaches and greater rigor of conceptual analysis.

Management of Leptoglossus occidentalis Heidemann in lodgepole pine (Pinus contorta var. latifolia Engelm.) seed orchards of British Columbia, Canada, would be improved with knowledge of its damage potential at different times of the growing season. Mesh insect-exclusion bags were placed over cones, and adults or nymphs of L. occidentalis were enclosed in different bags for 9 periods of 10 to 38 days between 6 May and 17 September 2004. Feeding by adult females between 6 May and 28 May reduced total extractable seeds, a result of ovule damage before fertilization. Reduction in the number of filled seeds per cone was highest between 6 May and 29 June, with each adult female reducing yield by approximately 1.7 seeds per day. Between 29 June and 10 August, each adult female reduced the number of filled seeds per cone (seed set) by 1.0 to 1.25 per day. Seed set reduction declined to approximately 0.25 filled seeds per adult female per day after 10 August. Third to fifth instars caused seed set reduction between 0.6 and 1.2 filled seeds per cone per nymph feeding day from 29 June to 10 August. Utilizing these findings would improve management of L. occidentalis in a commercial seed orchard setting.

The butterfly Pieris napi (L.) and relatives exemplify recently evolving taxa, exhibiting variation that makes their evolutionary dynamics interesting, but their systematics difficult. Wing-pattern characters commonly used to distinguish these Holarctic insects display both genetic polymorphism and environmentally-cued polyphenism. Often, these causes of variation are confounded, impairing the characters’ phylogenetic usefulness. DNA sequences of four mitochondrial genes offer an independent view of pierine phylogeny. Sampling diverse relatives within family Pieridae assists resolution of the P. napi complex, suggests previous underestimation of clade diversity in subfamily Pierinae, and shows that other genera near Pieris also display confusions of wing-pattern-based phylogenetic inference. The European P. napi is sister to all North American taxa, and is well diverged from them all in sequences. The North American taxa comprise a northern subclade including Pieris oleracea, and questionably distinct Pieris‘angelika’, and a southern subclade including distinct Pieris virginiensis, Pieris marginalis, and Pieris macdunnoughii, and other regional entities yet to be clarified. Weak bootstrap support for some nodes in this group arises from a closeness of sequence identity rather than character conflict; more sequence data and denser geographical sampling may resolve these nodes more clearly. Evidence of reproductive isolation, from other experimental hybridization studies, agrees with the DNA results where these conflict with other divergent character sets. The system offers much promise for a deeper understanding of character evolution in relation to phyletic differentiation. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 413–435.

Genotype–phenotype–environment interactions in temperate-zone species of Colias Fabricius, 1807 have been well studied in evolutionary terms. Arctic and alpine habitats present a different range of ecological, especially thermal, conditions under which such work could be extended across species and higher clades. To this end, we survey variation in three genes that code for phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), and glucose-6-phosphate dehydrogenase (G6PD) in seven arctic and alpine Colias taxa (one only for G6PD). These genes are highly polymorphic in all taxa studied. Patterns of variation for the PGI gene in these northern taxa suggest that the balancing selection seen at this gene in temperate-zone taxa may extend throughout northern North America. Comparative study of these taxa may thus give insight into the mechanisms driving genetic differentiation among subspecies, species, and broader clades, supporting the study of both micro- and macro-evolutionary questions.

Using oral swab samples to detect West Nile virus in dead birds, we compared the Rapid Analyte Measurement Platform (RAMP) assay with VecTest and real-time reverse-transcriptase-polymerase chain reaction. The sensitivities of RAMP and VecTest for testing corvid species were 91.0% and 82.1%, respectively.

The sex pheromone of the fir coneworm moth consists of a blend of (3Z,6Z,9Z,12Z,15Z)-pentacosapentaene and (9Z, 11E)-tetradecadienyl acetate. Analogous blends of polyunsaturated, long-chain hydrocarbons with much shorter chain aldehydes or alcohols recently have been discovered in three other moth species in the superfamily Pyraloidea. These combinations of components from two distinct structural classes may represent an important and widespread new pheromone blend motif within the Lepidoptera.

The VecTest WNV assay is adequate for diagnostic and surveillance purposes in American Crows, Blue Jays, and House Sparrows., The VecTest antigen-capture assay for West Nile virus was performed on oral and tissue swabs from dead birds in New York State from April 2003 through July 2004. Results were compared with those from real-time reverse transcriptase–polymerase chain reaction of kidney or brain. Oral VecTest sensitivity is adequate for surveillance in American Crows (Corvus brachyrhynchos) (87%), Blue Jays (Cyanocitta cristata) (80%), and House Sparrows (Passer domesticus) (76%). Oral VecTest performed well for small samples of American Kestrels (Falco sparverius), Northern Cardinals (Cardinalis cardinalis), Common Grackles (Quiscalus quiscula), and House Finches (Carpodacus mexicanus). Poor sensitivity occurred in most raptors, Mourning Doves (Zenaida macroura), Fish Crows (Corvus ossifragus), and American Robins (Turdus migratorius). Specificity was excellent (98%), except for false-positive results that occurred mostly in Gray Catbirds (Dumatella carolinensis), Green Herons (Butorides virescens), and tests of blood and tissues. Feather pulp and kidney may be useful for VecTest assays in corvids.

Presently, in USA there is no domestic crop source of oil rich in medium chain fatty acids (MCFAs) (i.e., oils composed of triglycerides with fatty acid chains between 8 and 14 carbons long). Several species of the genus Cuphea (Lythraceae) accumulate saturated MCFAs in their seed-storage lipids, and some species grow well in short-season temperate climates. Recent efforts have been successful in semi-domesticating genotypes of Cuphea for crop production. However, little is known about best management practices for production. A 2-year study was conducted in west central Minnesota, USA, to determine optimum inter-row spacing for row culture production of Cuphea. Seed was sown in inter-row spacings of 0.125, 0.25, 0.375 and 0.50 m in 1999, and a fifth spacing of 0.75 m was added in 2000. In 2000, seed yield averaged nearly 1000 kg ha−1, which was about 40% greater than in 1999. Seed yields were not significantly affected by row spacing. Plants in wider rows compensated for yield by producing more branches and seed pods per plant. The number of filled pods per plant was as much as 70% greater for plants in the widest as compared to the narrowest row spacing. Data indicate that this was due in part to competition among plants for available light and nutrient resources. Row culture of Cuphea in the northern Corn Belt of the US appears favorable. However, due to its indeterminate habit, growth and yield of Cuphea in row culture may be more responsive to plant population density than inter-row spacing.

We report that a chemical stimulus from a herbivore, a galling insect, changes plant morphology and physiology to benefit the herbivore. Previous studies could not determine whether insect galls are induced by mechanical or chemical stimuli because feeding and oviposition both occurred at the site of gall formation. We report that the mouthparts of a spruce-galling insect, Adelges cooleyi, were inserted in stem phloem cells far from induced galls, that tissues between mouthparts and galls appeared normal, and that the ability to initiate galls was inversely correlated with distance from buds (potential gall sites). Thus the effects of chemical stimuli were unambiguously separated from any mechanical influence of probing stylets or ovipositors. Our results strongly suggest that galls were induced by a chemical stimulus transported to buds via vascular tissue and that its efficacy was dose-dependent.

Leptoglossus occidentalis Heidemann is a serious pest of second-year cones in conifer seed orchards. We investigated the impact of this insect on first-year pine conelets. L. occidentalis adults and nymphs were caged on conelets of lodgepole pine, Pinus contorta var. latifolia Engelmann, and western white pine, Pinus monticola Dougl. ex D. Don. Abortion in lodgepole pine conelets was negligible, but feeding by nymphs reduced seed production by 75% compared with unexposed controls. Mortality of nymphs on lodgepole pine conelets was almost 100%, suggesting that conelets of this species are not a suitable food source for L. occidentalis. When nymphs had access to second-year cones, seed set in conelets was unaffected, and survival of nymphs greatly improved. Seventy-five percent of western white pine conelets exposed to L. occidentalis nymphs aborted, compared with none in unexposed controls. Seed set in surviving cones was reduced by 47%. Conelet abortion fell to 10% when nymphs were provided with an alternative food source. In pine seed orchards, damage to conelets probably is caused primarily by nymphs remaining on trees after second-year cones have been harvested.

Select germplasm of Cuphea, developed from an interspecific hybridization of C. viscosissima Jacq. and C. lanceolata f. Silenoides W.T. Aiton, shows good potential for commercial production in short-season temperate climates. Cuphea seed oil could serve as a domestic source of medium-chain fatty acids, which are in high demand by the chemical manufacturing industry. However, little is known about proper management practices for Cuphea agronomic production. A field study was conducted in west central Minnesota to determine optimum time of sowing in the northern Corn Belt region and describe influences of sowing date on growth and seed yield components of a semidomesticated germplasm line (PSR23). Seed was sown on 15 April, 1 May, 15 May, 1 June, and 15 June 1999 and 2000. Sowing in May resulted in greatest seed and seed-oil yields, which were as high as 1.09 and 0.29 Mg ha -1 , respectively. Seed yield declined as much as 31% when sown 15 April and 65% when sowing was delayed until 15 June. Plants developed from seed sown before June tended to form more branches and accumulated a greater amount of aboveground biomass. Typically, there was a distinct decrease in plant biomass accumulation and most seed yield components when sowing date was delayed until June. Cuphea PSR23 can be successfully grown in the northern Corn Belt, with early to mid May being the best time for sowing.

Postzygotic isolation between incipient species results from the accumulation of incompatibilities that arise as a consequence of genetic divergence. When phenotypes are determined by regulatory interactions, hybrid incompatibility can evolve even as a consequence of parallel adaptation in parental populations because interacting genes can produce the same phenotype through incompatible allelic combinations. We explore the evolutionary conditions that promote and constrain hybrid incompatibility in regulatory networks using a bioenergetic model (combining thermodynamics and kinetics) of transcriptional regulation, considering the bioenergetic basis of molecular interactions between transcription factors (TFs) and their binding sites. The bioenergetic parameters consider the free energy of formation of the bond between the TF and its binding site and the availability of TFs in the intracellular environment. Together these determine fractional occupancy of the TF on the promoter site, the degree of subsequent gene expression and in diploids, and the degree of dominance among allelic interactions. This results in a sigmoid genotype–phenotype map and fitness landscape, with the details of the shape determining the degree of bioenergetic evolutionary constraint on hybrid incompatibility. Using individual-based simulations, we subjected two allopatric populations to parallel directional or stabilizing selection. Misregulation of hybrid gene expression occurred under either type of selection, although it evolved faster under directional selection. Under directional selection, the extent of hybrid incompatibility increased with the slope of the genotype–phenotype map near the derived parental expression level. Under stabilizing selection, hybrid incompatibility arose from compensatory mutations and was greater when the bioenergetic properties of the interaction caused the space of nearly neutral genotypes around the stable expression level to be wide. F2’s showed higher hybrid incompatibility than F1’s to the extent that the bioenergetic properties favored dominant regulatory interactions. The present model is a mechanistically explicit case of the Bateson–Dobzhansky–Muller model, connecting environmental selective pressure to hybrid incompatibility through the molecular mechanism of regulatory divergence. The bioenergetic parameters that determine expression represent measurable properties of transcriptional regulation, providing a predictive framework for empirical studies of how phenotypic evolution results in epistatic incompatibility at the molecular level in hybrids.

Leptoglossus occidentalis Heidemann (Heteroptera: Coreidae) is a North American conifer seed pest that was accidentally introduced to Europe. In the Mediterranean area, it threatens the production of Pinus pinea Linna- eus seeds. The egg-parasitoid Gryon pennsylvanicum (Ashmead) (Hymenop- tera: Platygastridae), the main natural enemy in the native range of L. occidentalis, was imported from British Columbia to Italy. Pre-release risk assessments were made under quarantine conditions by no-choice tests conducted with na€ıve and experienced G. pennsylvanicum offering single eggs of target and non-target species for varying exposure times (1, 4, 48 h). G. pennsylvanicum successfully parasitized from 75% to 100% of the target host eggs. Only one female specimen of the egg-parasitoid emerged from a non-target egg (Gonocerus juniperi Herrich-Schaeffer, He- teroptera: Coreidae). Two dead female specimens were found, one inside an egg of Coreus marginatus (Linnaeus) (Heteroptera: Coreidae) and one in an egg of Camptopus lateralis (Germar) (Heteroptera: Alydidae). All three cases occurred at the longest oviposition exposure time. Results obtained with this conservative approach suggest that the risk to non-target species of releasing G. pennsylvanicum in Italy is low.

La faible grenaison est un probleme serieux dans les plantations de production de graines du pin de Murray (Pinus contorta var. latifolia Engelmann) dans la region interieure du sud de la Colombie-Britannique. Nous avons eprouve l'hypothese selon laquelle le responsable de cette reduction est Leptoglossus occidentalis Heidemann. Au cours d'une experience sur la pollinisation, des cones enfermes dans des sacs a l'epreuve des insectes ont produit plus de graines pleines par cone que les cones non proteges. Dans une autre experience d'ensachage, les cones qui ont ete enfermes en presence d'une femelle de L. occidentalis avec sa progeniture n'ont produit grosso modo qu'une graine pleine par cone, alors que les cones temoins non ensaches produisent environ 28 graines pleines par cone. Les changements de microclimat associes a l'ensachage ne semblent pas responsables de l'augmentation de la grenaison observee dans les cones mis en sac. Leptoglossus occidentalis a egalement ete exclus des arbres par un traitement a l'insecticide fenvalerate. Les cones des arbres traites au fenvalerate ont produit plus de 11 graines pleines par cone, alors que les cones des arbres temoins traites a l'eau ont donne moins de 1,7 graine pleine par cone. Ces donnees indiquent que L. occidentalis doit etre considere comme une espece tres nuisible dans les plantations de production de graines du pin de Murray.

Diagnostic and analytical findings are presented for 105 common loons (Gavia immer) found dead or debilitated in New York (USA) from 1972-99. Aspergillosis (23% of cases) and ingestion of lead fishing weights (21%) were the most common pathologies encountered. Stranding on land, shooting, other trauma, gill nets, air sacculitis and peritonitis, and emaciation of uncertain etiology accounted for most of the remaining causes of disease or death. Analysis for total mercury in the liver of 83 loons yielded a geometric mean (gm) of 10.3 mg/kg (wet basis) and range of 0.07 to 371 mg/kg, with emaciated birds generally showing higher levels. Organochlorine contaminant levels in brain were generally low, principally consisting of PCB's (gm = 2.02 mg/kg) and DDE (0.47 mg/kg).

▪ Abstract Knowledge of both prokaryotic and eukaryotic organisms is essential to the study of molecular evolution. Their common ancestry mandates that their molecular functions share many aspects of adaptation and constraint, yet their differences in size, ploidy, and structural complexity also give rise to divergent evolutionary options. We explore the interplay of adaptation, constraint, and neutrality in their evolution by the use of genetic variants to probe molecular function in context of molecular structure, metabolic organization, and phenotype-environment interactions. Case studies ranging from bacteria to butterflies, flies, and vertebrates emphasize, among other points: a. the importance of moving from initial recording of evolutionary pattern variation to studying the processes underlying the patterns, by experiment, reconstructive inference, or both; b. the complementarity, not conflict, of finding different performance and fitness impacts of natural variants in prokaryotes or eukaryotes, depending on the nature and magnitude of the variants, their locations and roles in pathways, the nature of molecular function affected, and the resulting organismal phenotype-environment interactions leading to selection or its absence; c. the importance of adaptive functional interaction of different kinds of variants, as in gene expression variants versus variants altering polypeptide properties, or interaction of changes in enzymes' active sites with complementary changes elsewhere that adjust catalytic function in different ways, or coadaptation of different steps' properties in pathways; d. the power afforded by combining structural and functional analyses of variants with study of the variants' phenotype-environment interactions to understand how molecular changes affect (or fail to affect) adaptive mechanisms “in the wild.” Comparative study of prokaryotes and eukaryotes in this multifaceted way promises to deliver both new insights into evolution and a host of new and productive questions about it.

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows ( Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo ( Phoenicopterus chilensis ), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein–specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

We study here the connections among body temperature variation, flight performance and flight ‘fuel’ metabolism in Colias eurytheme butterflies, to begin re-examining the metabolic reasons for animal thermoregulation. Methods are presented for (a) stable extraction of adenylates (and other metabolites) from the flight muscles of individual Colias eurytheme, (b) automated separation and quantitative analysis of individual adenylate samples by high-pressure liquid chromatography and (c) reliable, low-variance assay of inorganic phosphate levels in the same extracts. Correlations among metabolite concentrations and two indices of muscle cytosol ATP maintenance occur as expected on general metabolic principles. [ATP] and [ATP]/[ADP] decline from resting levels to reach a plateau in the first minute of free, interrupted flight, while [AMP] increases at the same time; these concentrations do not vary further for up to 6 min total flight time. In an initial test of the alternative metabolic bases of the thermoregulation of Colias eurytheme, we find that [ATP]/[ADP] rises between a body temperature, Tb, of 31 and 35 °C, at the base of the behavioral thermal optimum for flight, but then decreases again at Tb=39 °C, at the top of the behavioral thermal optimum and well short of damaging temperatures. This is not consistent with the view that metabolic effectiveness increases monotonically up to the lower limits of thermal damage to enzymes, but supports an alternative hypothesis that the narrowness of thermoregulation results from a system-based constraint on the breadth of temperature over which maximal energy processing is possible. ‘Data on the biochemistry of insect thermoregulation are extremely scanty.’ G. A. Bartholomew (1987)

In New York, an epizootic of American crow (Corvus brachyrhynchos) deaths from West Nile virus (WNV) infection occurred during winter 2004–2005, a cold season when mosquitoes are not active. Detection of WNV in feces collected at the roost suggests lateral transmission through contact or fecal contamination.

The sulfur butterflies, Colias spp., and their relatives in the family Pieridae have been the subjects of diverse behavioral, ecological, and evolutionary studies. However, their phylogeny is uncertain in many respects. We used DNA sequences from 2 mitochondrial gene blocks, 333 bp of the cytochrome oxidase I subunit (CO I) and 1,261 bp from the 2 ribosomal genes and the tRNA between them (rDNA), as character sources to test existing phylogenetic hypotheses and begin to infer others. The rDNA block resolves better at deeper nodes of the phylogeny, and the CO I block at shallower nodes. Our results support sister status for subfamilies Coliadinae and Pierinae within Pieridae; independent tribal status for Euchloini and Pierini within Pierinae; status as sister genera for Colias and Zerene within Coliadinae; and monophyly within subgenus C. (Eucolias) of all North American Colias studied. Our results suggest that the Neotropical coliad genus Eurema may warrant splitting, as some early workers proposed, but do not support the recently proposed splitting of Eurasian C. erate from subgenus C. (Eriocolias) into the separate subgenus C. (Neocolias) . (Lepidoptera: Pieridae)

Three quantitative methods (Taylor, Iwao, and binomial) were used to estimate spatial aggregation patterns and refugia of the spider mite prey, Tetranychus urticae Koch and the phytoseiid mite predator Neoseiulus fallacis (Garman) on hop relative to the time of year, plant height, and presence or absence of predators. Using the Taylor methods, there was little change in aggregation of T. urticae with plant height, whether predators were released or not, and only a small change with season. The Iwao parameters indicated that spider mite colonies became larger but less clumped through the season and that in the presence of predators, colony size decreased and patchiness increased. The seasonal change in predator distribution as measured by the Iwao parameters closely reflected prey distributions with clump size increasing and clumps becoming less aggregated. Binomial data showed there was a refugium of hop leaves in which T. urticae did not occur and a refugium of spider mites colonies without predators. These refugia probably result from the combined effects of rapid growth of the hop plant and a spatial incongruence of predator and prey. The refugia may add to stability and persistence of predators and prey on hops. Implications of spatial patterns and refugia to biological control and sampling are discussed.

International audience; Combining pheromone trapping and genetic analyses can be useful when trying to resolve complexes of closely related insect taxa that are difficult to distinguish based on morphological characters. Nearctic and Palearctic populations of the spruce seed moth, Cydia strobilella L., have been considered taxonomically synonymous since 1983, but more recent work revealing distinct sex pheromones for Canadian and Swedish moths suggest that populations in the two regions belong to different species. In order to test this hypothesis, we performed field trapping using different pheromone lures at ten sites in North America, Europe and Asia, and reconstructed phylogenetic relationships among trapped moths using mitochondrial (cytochrome oxidase subunit I) and nuclear (elongation factor 1 alpha) DNA sequence data. Trapping data and tree topologies for both genes revealed distinct pherotypes in North America and Eurasia. A genetically distinct population from China was investigated further with respect to its sex pheromone. Electrophysiological data indicated that Chinese females produce a deviant ratio of the sex pheromone components (dienic acetates) compared to Swedish females. However, trapping experiments in both areas revealed a similar broad response profile in males to a wide range of acetate ratios, and these populations should be considered taxonomically synonymous. A previous suggestion of an agonistic effect on the attraction of C. strobilella males in Sweden when adding the corresponding alcohols to the binary acetate blend was also tested in Sweden as well as in China, with no observed effect on attraction of males. In conclusion, our study demonstrates the great potential of using pheromone trapping as a tool for identification and delimitation of taxa within cryptic species complexes. Based on our data, Nearctic and Palearctic populations of C. strobilella should be considered different species, and C. youngana Kearfott stat. rev. is resurrected here as valid name for North American populations, which was the case before the revision in 1983.

In lowland Cobs butterflies, genotypes of the enzyme phosphoglucose isomerase (PGI) show major differences in molecular function, from which genotypic differences in organismal performance and fitness components in the wild are accurately predictable. The alpine species Colias meadii seems to share electromorph alleles with lowland congeners at PGI and phosphoglucomutase (PGM). However, high-resolution electrophoresis finds differences between PGI electromorphs of meudii and those of lowland taxa. Common C. meadii genotypes differ in thermal stability and are less thermally stable than similar electromorph genotypes in lowland Colius eurytheme. These meudii genotypes show heterozygote advantage in the kinetic parameters K,,, and V,JK,,, (and differ sharply from genotypes of C. eurytheme). The thermally more stable homozygote is the kinetically less effective one, extending the tradeoff of kinetics vs. stability in PGI homozygotes, seen in lowland taxa, to C. meudii. Positive evidence is given for the absence of assortative mating and segregation distortion at both PGI and PGM. The functional differences among PGI genotypes explain previously observed heterozygote advantage in flight capacity and survivorship and correctly predict heterozygote advantage in male mating success, of C. meudii’s PGI genotypes. Though functional information is not yet available on C. meudii PGM variants, these also show heterozygote advantage in male mating success but do not interact with PGI. Thus, differences in molecular function result in fitness component differences among PGI allozymes in alpine Cobs, as well as in lowland ones. This is the more remarkable because the parallelism is not based on allelic identity. These results support expectations of evolutionary bioenergetics and emphasize the hazards of using ordinary electrophoresis to infer allozymes’ identity among taxa. In alternative scenarios for Colius’ PGI evolutiondivergence from ancestral polymorphism, or independent origin-these results suggest major constraint, based in protein structure, on ability of PGI alleles to maximize fitness-related biochemical performance when homozygous.

Biological control of the twospotted spider mite, Tetranychus urticae Koch, on hops, Humulus lupulus L., with the predator Neoseiulus fallacis (Garman) was explored to understand the factors important in successful T. urticae management. The timing of introductions of N. fallacis , the height of release on the plant, overwinter survival, and cultural factors were investigated to determine the optimal use patterns for this predator. Release height seemed to be unimportant, but release timing was influential when there were unusually low or high T. urticae densities. N. fallacis overwinters in hop fields, but early spring cultural practices inhibit survival to summer. Three cultural practices, all for reduction of foliar growth, were found to be detrimental to biological control. Predators released into 0.25-ha plots in commercial fields maintained T. urticae to commercially acceptable levels throughout the season. A provisional management protocol is described for biological control of T. urticae with N. fallacis .

Biological control of Tetranychus urticae Koch was studied on hops, Humulus lupulus L., a rapidly growing perennial crop, in westem Oregon. Single or mixed species of Neoseiulus fallacis (Garman), Metaseiuius occidentalis (Nesbitt), Typhlodromus pyri Scheuten, and Amblyseius andersoni Chant were released inoculatively into individual plants in 1992–1993. Success of T. urticae control depended on the phytoseiid species released and the ratios and distributions of pest and predaceous mites. The most effective control was by N. fallacis or M. occidentalis or a mixture of both species. In 1993, M. occidentalis provided poor control, probably because of cool weather. T. pyri and A. andersoni provided some control, but always less than N. fallacis and M. occidentalis . Analysis of spatial distributions indicated that both N. fallacis and M. occidentalis dispersed throughout hop plants if T. urticae were not abundant, but they stayed on the lower parts of the plant when T. urticae were abundant. Factors governing the distributions and dispersal of T. urticae and phytoseiids on hops and the effectiveness of biological control are discussed.

The lethal humidity (LH50) responses at 20°C of eggs of two strains of Neoseiulus fallacis (Garman) were 71.6 and 69.7%; of three strains of Amblyseius andersoni (Chant) were 62.9, 62.0 and 62.4% and of one strain each of Typhlodromus pyri Scheuten and Metaseiulus occidentalis Nesbitt were 55.0 and 28.4%, respectively. Eggs of three genetically distinct strains of A. andersoni from Oregon, the Netherlands and Italy did not respond differently from one another nor did eggs of freely hybridizing N. fallacis from Michigan and Oregon. Mortality of larvae through development to early protonymphs at 50% RH, 20°C. was 91.9, 82.3, 46.2 and 31.0% for fed mites and 98.1, 83.2, 67.0 and 89.7% for unfed mites of Oregon strains of N. fallacis, A. andersoni, T. pyri and M. occidentalis, repectively. Fed larvae-protonymphs of M. occidentalis and T. pyri were more tolerant of low humidity than fed larvae-protonymphs of N. fallacis and A. andersoni. Mortality was less for fed than unfed larvae-protonymphs of M. occidentalis and T. pyri, but there were no differences for A. andersoni and N. fallacis. Levels of feeding by predator larvae on T. urticae and cannibalism by phytoseiid protonymphs contributed to species differences. Responses to humidity are discussed in relation to geographic and host plant distributions and biological control by single or mixed species populations of phytoseiids.

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum . Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.

BackgroundMassive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS). A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals.Methodology/principal findingsWe have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii) G. destructans DNA was directly amplified from infected bat tissues, (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv) RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans.Conclusions/significanceFurther studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat-fungus relationships, and should aid in the screening of biological and chemical control agents.

A molecular evolutionary explanation of natural genetic variation requires analysis of specific variants’ evolutionary dynamics. To pursue this for phosphoglucose isomerase (PGI) of Colias butterflies, whose polymorphism is maintained by strong natural selection, we assembled a large data set of wild haplotypes, highly variable at the amino acid and DNA levels. The most common electrophoretic, i.e., charge macrostate, allele class, 3, is conserved in its pattern of charged amino acid residues. The next most common macrostate, 4, has multiple patterns of charge, i.e., microstates, while less common (1, 2, 5, 6) macrostates are very diverse. Macrostate 4 shows significant linkage disequilibrium (LD) among its variants, especially for two groups of five haplotypes each. We find extensive intragenic recombination among all haplotypes except the two high-LD groups of macrostate 4, which display none. Phyletic relations among haplotypes are largely reticulate, again except for the high-LD groups of macrostate 4, which form clades with strong bootstrap support. Charge-changing and linked charge-neutral amino acid variants occur in diverse parts of PGI’s sequence. Homology-based modeling of PGI’s structure shows that these regions are related spatially in ways suggesting functional interaction. The high-LD groups of macrostate 4 display parallel amino acid variation in these regions. This pattern of haplotype clades with high LD among multiple varying sites, emerging from chaotically recombining variation, may be a “signature” of refinement of complex adaptive sequences by recombination and selection. It should be tested further in this study system and others as a possibly general feature of the evolution of living complexity.

Colias eurytheme butterflies display extensive allozyme polymorphism in the enzyme phosphoglucose isomerase (PGI). Earlier studies on biochemical and fitness effects of these genotypes found evidence of strong natural selection maintaining this polymorphism in the wild. Here we analyze the molecular features of this polymorphism by sequencing multiple alleles and modeling their structures. PGI is a dimer with rotational symmetry. Each monomer provides a critical residue to the other monomer's catalytic center. Sequenced alleles differ at multiple amino acid positions, including cryptic charge-neutral variation, but most consistent differences among the electromorph alleles are at the charge-changing amino acid sites. Principal candidate sites of selection, identified by structural and functional analyses and by their variants' population frequencies, occur in interpenetrating loops across the interface between monomers, where they may alter subunit interactions and catalytic center geometry. Comparison to a second (and basal) species, Colias meadii, also polymorphic for PGI under natural selection, reveals one fixed amino acid difference between their PGIs, which is located in the interpenetrating loop and accompanies functional differences among their variants. We also study nucleotide variability among the PGI alleles, comparing these data to similar data from another glycolytic enzyme gene, glyceraldehyde-3-phosphate dehydrogenase. Despite extensive nonsynonymous and synonymous polymorphism at PGI in each species, the only base changes fixed between species are the two causing the amino acid replacement; this absence of synonymous fixation yields a significant McDonald-Kreitman test. Analyses of these data suggest historical population expansion. Positive peaks of Tajima's D statistic, representing regions of neutral "hitchhiking," are found around the principal candidate sites of selection. This study provides novel views of molecular-structural mechanisms, and beginnings of historical evidence, for a long-persistent balanced enzyme polymorphism at PGI in these and perhaps other species.