Components of morphological variation in Baikalian endemial cyclopid Acanthocyclops signifer complex from different localities

Use of traditional methods for morphological studies only permits the analysis of a small part of the information embodied in morphological structures. Besides comparing populations using the mean values of characters which allows one to estimate their morphological similarity, analysis of variation among individuals within a population can be informative. Variation among individuals consists of factorial and stochastic components. The factorial component is an upper estimate of genetic heterogeneity and thus permits one to evaluate the population's adaptability. The stochastic component (estimated by fluctuating asymmetry, i.e. random deviations from perfect bilateral symmetry), being a measure of developmental stability, is an indicator of a population's fitness. Assessment of measurement error is necessary for assessment of the true value of the stochastic component and for selection of the most informative characters. Such analysis allows one to extract additional information from morphological data in comparison with methods traditionally used on copepods. This approach was applied to an analysis of morphological variation in the study of the Baikalian endemic cyclopoid Acanthocyclops signifer (Mazepova) from three different isolated localities. Characters typically used in studies of taxonomy of this group are considered here. Measurement error was rather high (more than 50% of the stochastic component), which can be explained by technical difficulties of measuring the characters. All populations differ in the mean values of the characters. This shows the taxonomic heterogeneity of this group and reveals the necessity of its taxonomic revision. Populations also differ in the level of stochastic and factorial components of the total variance. The data are interpreted from the point of view of taxonomy and the possible evolution of the group.     

Genetic variation in the life‐history traits of Epiphyas postvittana: population structure and local adaptation

The light brown apple moth, Epiphyas postvittana (Walker) shows high intraspecific variability in morphological, physiological, demographic and behavioural characters. To gain insight into the extent of adaptation and evolutionary changes in response to environmental heterogeneity in this species, quantitative genetic analyses of life‐history variation were conducted for two natural populations under two thermal conditions (23°C and 28°C). Paternal half‐sib heritability and genetic correlation in six life‐history traits (i.e. development time, adult body weight, adult lifespan, age at first reproduction, the number of eggs laid during the first 5 days after emergence, and total fecundity) were compared. Significant heritabilities were shown consistently in development time; this is also true for adult body weight, except for the Canberra population at 23°C. However, neither population differences nor the effect of temperature were statistically detectable for any of these heritabilities, confirming the genetically determined flexibility. Positive genetic correlations between development time and adult body weight, and negative genetic correlations between the number of eggs laid during the first 5 days and adult lifespan were present for these populations at both temperatures, indicating the presence of genetic constraints. Pairwise comparisons of genetic correlations revealed the heterogeneity of the two populations and across temperatures. These results suggest that the structure of genetic covariance might have changed significantly during the divergence of natural populations and in response to the alteration of environmental conditions in E. postvittana.     

Variations in morphological and life-history traits under extreme temperatures in <Emphasis Type="Italic">Drosophila ananassae</Emphasis>

Using half-sib analysis, we analysed the consequences of extreme rearing temperatures on genetic and phenotypic variations in the morphological and life-history traits of Drosophila ananassae. Paternal half-sib covariance contains a relatively small proportion of the epistatic variance and lacks the dominance variance and variance due to maternal effect, which provides more reliable estimates of additive genetic variance. Experiments were performed on a mass culture population of D. ananassae collected from Kanniyakumari (India). Two extremely stressful temperatures (18°C and 32°C) and one standard temperature (25°C) were used to examine the effect of stressful and non-stressful environments on the morphological and life-history traits in males and females. Mean values of various morphological traits differed significantly among different temperature regimens in both males and females. Rearing at 18°C and 32°C resulted in decreased thorax length, wing-to-thorax (w/t) ratio, sternopleural bristle number, ovariole number, sex comb-tooth number and testis length. Phenotypic variances increased under stressful temperatures in comparison with non-stressful temperatures. Heritability and evolvability based on among-sires (males), among-dams (females), and the sum of the two components (sire + dam) showed higher values at both the stressful temperatures than at the non-stressful temperature. These differences reflect changes in additive genetic variance. Viability was greater at the high than the low extreme temperature. As viability is an indicator of stress, we can assume that stress was greater at 18°C than at 32°C in D. ananassae. The genetic variations for all the quantitative and life-history traits were higher at low temperature. Variation in sexual traits was more pronounced as compared with other morphometric traits, which shows that sexual traits are more prone to thermal stress. Our results agree with the hypothesis that genetic variation is increased in stressful environments.     

Effect of larval rearing temperature on adult female morphology of Anopheles (Nyssorhynchus) albimanus (Diptera: Culicidae)

Summary

Time to pupation, percent survival to pupation, and percent adult emergence of Anopheles albimanus Wiedemann decreased at higher larval rearing temperature. Mosquitoes reared at 30°C experienced higher mortality during the pupal stage than did mosquitoes reared at 22°C. Analysis of variance revealed that wing length and costal wing spot patterns of adult female A. albimanus were affected by larval rearing temperature. Female A. albimanus reared at 22°C had longer wings, and larger basal pale + prehumeral pale, prehumeral dark, and humeral pale costal wing spots than did female siblings reared at 30°C. Female A. albimanus reared at 30°C had larger subcostal pale spots than did female siblings reared at 22°C. Analyses of 2x2 contingency tables indicated that sex ratio was independent of larval rearing temperature, whereas survival to the adult stage and coalescence of wing spots were not independent of rearing temperature. The need to examine stability of morphological characters under differing environmental conditions is discussed.     

Effect of temperature on the walking behaviour of an egg parasitoid: disentangling kinetic response from integrated response

1. This study investigated the effect of temperatures ranging from 10.8 to 34.2 °C on seven walking parameters of an egg parasitoid, Anaphes listronoti. Those responses were compared with a theoretical kinetic model in order to disentangle the kinetic response of the insect from its integrated response. 2. Walking speed increased continuously with temperature, but walking distance and duration were maximised between 25 and 30 °C, and decreased at higher and lower temperatures. At the lowest temperature, females unexpectedly walked a greater distance and for a longer duration in comparison with intermediate temperatures. 3. The number of walking bouts followed the same polynomial trend as walking distance and duration, with a maximum between 30 and 35 °C. The duration of walking bouts was maximised at 20 °C and decreased at lower and higher temperatures, whereas the duration of resting bouts linearly decreased with increasing temperature. There was no effect of temperature on the turning rate of females. 4. For A. listronoti, walking speed and duration of resting bouts followed the kinetic response, but the other behavioural components did not, especially at the lowest and highest temperatures. Walking distance and duration were higher than expected under the kinetic response at 10.8 °C and lower than expected at 34.2 °C. This pattern suggests an integrated response combining behavioural escape from adverse temperature and energy saving. 5. This detailed analysis of the walking behaviour of A. listronoti illustrates the complexity of insect behavioural responses to temperature and the difficulty involved in assessing underlying mechanisms.     

Morphological analysis of some cryptic species in the Acanthocyclops vernalis species complex from North America

Patterns of morphological variation and reproductive isolation were examined for several North American populations of copepods in the Acanthocyclops vernalis Fischer A., 1853 (Copepoda, Cyclopinae) species complex. The copepods were collected from six sites in Wisconsin, U.S.A. Morphological analysis of 120 adult females revealed that a character used previously to distinguish species in this group was unreliable because of phenotypic plasticity. Most of the morphological variance was due to environment (Laboratory vs. field) and to field site. Relatively little of the variation was due to measurement error or asymmetry. Multivariate ordination analysis produced poorly-defined clusters of individuals, suggesting that different biological species are difficult or impossible to distinguish using a set of easily-measurable morphological characters. In our study, morphological similarity was independent of geographic distance among sites, between 0.05 and 300 km. Isofemale lines within sites showed little or no reproductive isolation, but nearly complete isolation among sites. Reproductive isolation was also independent of morphology. These results suggest that the Acanthocyclops population at each site could be considered a distinct cryptic biological species. These copepods expressed morphological stasis – persistence of morphological uniformity despite reproductive isolation. Because of the effect of site and environment on morphology, we recommend using much larger collections (many sites), common garden experiments, and a multi-disciplinary approach (morphological, reproductive, chromosomal, and molecular) as the basis for future taxonomic research on putative copepod species.     

A cyanophycin synthetase from <Emphasis Type="Italic">Thermosynechococcus elongatus</Emphasis> BP-1 catalyzes primer-independent cyanophycin synthesis

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires l-aspartic acid (Asp), l-arginine (Arg), ATP, Mg²⁺, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg²⁺ or Mn²⁺ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 ± 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.     

A New Thermophilic Species of <Emphasis Type="Italic">Conidiobolus</Emphasis> from India

A Conidiobolus isolate growing optimally at 40°C was isolated from decomposing leaf litter and has been designated as a new species, Conidiobolus thermophilus. Colony growth, conidial discharge and smooth zygospore formation was rapid at 40°C, while comparative growth at 35 and 45°C was slower. On the basis of its thermophilic character and morphological distinctness from all other species, the isolate is considered as a species new to science. There have been no published reports of any thermophilic or thermotolerant strains of Conidiobolus. The present fungus was isolated as part of an ongoing programme of selective isolation of unusual/rare thermophilic fungi from compost and decomposed terrestrial plant materials.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

A COMPARISON OF TEMPERATURE-INDUCED POLYPHENISM IN AFRICAN BICYCLUS BUTTERFLIES FROM A SEASONAL SAVANNAH-RAINFOREST ECOTONE

Temperature-induced variation and norms of reaction have been analyzed for wing pattern elements of six species belonging to the African butterfly genus Bicyclus (Lepidoptera, Nymphalidae, Satyrinae). Five of these species are sympatric in Malawi and exhibit seasonal polyphenism in the savannah-rainforest ecotone. The sixth species originated from Cameroonian equatorial rainforest. The organisms were laboratory reared under four different temperature conditions ranging from 17–28°C. The variation in response to temperature is described by principal component analysis (PCA). Discrimination on the basis of plastic wing pattern characters was performed by discriminant function analysis (DFA) and unweighed pair-group method algorithm (UPGMA) clustering. A phylogenetic reconstruction based on adaptive plastic wing characters was compared with a cladogram built on “nonadaptive” characters. Results demonstrate that: (1) Phenotypic plasticity of wing pattern characters in response to temperature in laboratory-reared organisms is reminiscent of variation induced by seasonal change in the field. (2) Different wing pattern characters are under different control: “exposed” characters of butterflies at rest position are highly sensitive to temperature variation, whereas “hidden” characters, only visible during active behavior, are dominated by species differences. In general the sensitivity of the former can be attributed to their proposed function in deflecting predators. (3) The sexes differ especially in the size of those eyespots that are displayed during active behavior. (4) Species from seasonal and aseasonal environments react in a broadly similar manner to temperature variation. However, savannah species and species of aseasonal rainforest exhibit relatively shallow reaction norms, whereas reaction norms are steeper in species from the savannah-rainforest ecotone. Such a strong response was also apparent in so-called correlation networks between principal components for these species. (5) Phylogenetic distances are to some extent reflected in ordination in both PCA-space and DFA-space: closely related species of the safitza group remain close in both ordinations. The more distantly related species differ in ordination from a pattern as suggested by a phylogenetic reconstruction. It is argued that the wing pattern variation of these species reflects both adaptive processes and historical relationships.     

Effects of Temperature on the Antipredator Behaviour and on the Cholinergic Expression in the European Sea Bass (Dicentrarchus labrax L.) Juveniles

In this paper, an experimental approach was used to test for the parallel effects of temperature (T) increase on the antipredator behaviour and the cholinergic expression in the juvenile European sea bass (Dicentrarchus labrax L.). The effects of three T treatments (18, 22 and 26°C) were tested on the main behavioural components of the antipredator response towards live aquatic predators and aerial simulated attacks, whereas brain cholinergic expression was evaluated by choline acetyltransferase (ChAT) immunoblotting (Western blot) at the extreme values of the thermal range (18 and 26°C). Antipredator responses towards a live fish were analysed over pre‐exposure and exposure phases within a short temporal scale (20 s before and after the stimulus). The results suggest that T modulates several quantitative components of the antipredator behaviour. The mean shoaling index (shoal cohesiveness) was higher at 22°C than at 18 and 26°C during both the pre‐stimulus and the exposure phase. Conversely, the mean distances from the predator and the tank bottom were, respectively, lower and higher at 26°C than in the other two treatments. In regard to the antipredator response on the aerial stimulus, comparisons across treatments revealed statistically significant differences between fish performing freezing or latency to recovery, suggesting that the fright reaction has a higher persistence at the coldest T (18°C) than at 22 and 26°C. Western blot analysis revealed a reduction in brain ChAT expression in fish acclimated to 26°C compared to those at 18°C. Results were discussed in the light of the relationships between behavioural traits, metabolism and their consequences on the population level, as a response to climate change in coastal habitats.     

闽楠叶形态与叶脉网络性状关系对城市生长环境的响应

植物叶形态与叶脉网络功能性状的协同变异与权衡关系,对深入理解植物叶脉网络功能性状对环境变异的生态适应,以及预测植物物种生活习性对城市化过程的响应具有重要意义。闽楠作为珍贵的常绿阔叶树种,正在城市绿化中逐步推广。针对不同生长环境中（行道与植物园混交林）的闽楠,开展了叶形态与叶脉网络功能性状关系对城市生长环境的响应研究。研究结果表明：闽楠叶性状值基本满足正态分布,各性状变异系数保持在10%-20%之间,群体内性状变异较为丰富,单因素方差分析表明两种环境对叶形态性状的影响比叶脉网络系统的影响更明显;两种生长环境下闽楠叶形态性状组与叶脉网络功能性状组都具有极显著相关性,行道和植物园混交林典型性相关系数分别为0.804和0.795,叶形态性状与叶脉直径呈显著正相关,形态性状、叶脉直径与初级脉密度呈显著负相关;闽楠在响应城市生长环境的过程中呈现出相应的经济权衡机制,行道环境中闽楠以较大的初级脉密度和较小叶面积来确保水分获取和光合之间的平衡,植物园闽楠则采用较低初级叶脉密度、较高叶面积和叶脉直径的叶形态和叶脉网络构建模式。在选择闽楠作为城市绿化树种时,可将叶片形态性状组与叶脉网络功能性状组的协同变化和权衡关系作为选种依据,以提高闽楠在城市环境中的成活率和适应性。     

The evolution of social behaviour in Blaberid cockroaches with diverse habitats and social systems: phylogenetic analysis of behavioural sequences

The adequacy and utility of behavioural characters in phylogenetics is widely acknowledged, especially for stereotyped behaviours. However, the most common behaviours are not stereotyped, and these are usually seen as inappropriate or more difficult to analyze in a phylogenetic context. A few methods have been proposed to deal with such data, although they have never been tested on samples larger than six species, which limits their evolutionary interest. In the present study, we perform behavioural observations on 13 cockroach species and derive behavioural phylogenetic characters with the successive event‐pairing method. We combine these characters with morphological and molecular data (approximately 6800 bp) in a phylogenetic study of 41 species. We then reconstruct ancestral states of the behavioural data to study evolution of social behaviour in these insects with regard to their social systems (i.e. solitary, gregarious, and subsocial) and diversity of habitat choice. We report for the first time that nonstereotyped behavioural data are adequate for phylogenetic analyses: they are no more homoplastic than traditional data, and support several phylogenetic relationships that we discuss. From an evolutionary perspective, we show that the solitary species Thanatophyllum akinetum does not display original behavioural interactions, suggesting phylogenetic inertia of interactive behaviours despite a radical change in social structure. Conversely, the subsocial species Parasphaeria boleiriana shows original behavioural interactions, which could result from its peculiar social system or habitat. We conclude that phylogenetic approaches in studies of behaviour are useful for deciphering evolution of behaviour and discriminating between its different modalities, even for nonstereotyped characters. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 58–77.     

Prodigiosin-25 C

The equilibrium constant of the isomerization reaction between d-glucose and d-fructose which is catalyzed by a. glucose isomerase from Streptomyces sp. was obtained by both methods of chemical analysis and of kinetic study over the temperature range of 25° to 70°C.

It was found that the formation of d-fructose from d-glucose was an endothermic reaction with the heat of the reaction, ΔH, of +2220 cal/mole. The standard free energy change, ΔG, and the standard entropy change, ΔS, associated with the isomeric change were found to be +180 cal/mole and + 6.8 cal/deg. mole at 25°C, respectively. The values of these thermodynamic quantities at other temperature are also summarized.     

The Acclimation of European Sea Bass (Dicentrarchus labrax) to Temperature: Behavioural and Neurochemical Responses

Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28°C). After 21 d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28°C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28°C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22°C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28°C in comparison with those maintained at 18°C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28°C than at 18°C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass.     

Phenotypic plasticity of thermal tolerance contributes to the invasion potential of Mediterranean fruit flies (Ceratitis capitata)

1. The invasion success of Ceratitis capitata probably stems from physiological, morphological, and behavioural adaptations that enable them to survive in different habitats. However, it is generally poorly understood if variation in acute thermal tolerance and its phenotypic plasticity might be important in facilitating survival of C. capitata upon introduction to novel environments. 2. Here, by comparison of widely distributed C. capitata with a narrowly distributed congener, C. rosa, we show that both species have similar levels of survival to acute high and low temperature exposures under common rearing conditions. However, these species differ dramatically in the time‐course of plastic responses to acute low temperature treatments. 3. The range of temperatures that induce rapid cold hardening (RCH) are similar for both species. However, C. capitata has two distinct advantages over C. rosa. First, at 5°C C. capitata develops RCH significantly faster than C. rosa. Second, C. capitata maintains a RCH response longer than C. rosa (8 vs. 0.5 h). 4. A simple population survival model, based on the estimated time‐course of RCH responses determined for both species, was undertaken to simulate time to extinction for both species introduced into a similar thermally variable environment. The model showed that time to extinction is greater for C. capitata than for C. rosa, especially in habitats where temperatures frequently drop below 10°C. 5. Thus, variation in RCH responses may translate into significant variation in survival upon introduction to novel thermal habitats for C. capitata, particularly in cooler and more thermally variable geographic regions, and may contribute to their ongoing invasion success relative to other, more geographically constrained Ceratitis species.     

Factors influencing the radiative surface temperature of grey seal (<Emphasis Type="Italic">Halichoerus grypus</Emphasis>) pups during early and late lactation

The aim of this study was to examine the variation in body surface temperature of grey seal (Halichoerus grypus) pups throughout lactation in response to different environmental conditions. Radiative surface temperatures (T _r, °C) of pups were measured on the Isle of May (56°11′N, 02°33′W), southeast Scotland from 29 October to 25 November 2003. Records were obtained from a total of 60 pups (32 female and 28 male) from three different pupping sites during early and late lactation. Pups were sheltered from high wind speeds but air temperature, humidity and solar radiation at pupping sites were similar to general meteorological conditions. The mean T _r of all pups was 15.8°C (range 7.7–29.7°C) at an average air temperature of 10.2°C (range 6.5–13.8°C). There was no difference in the mean T _r of pups between early and late lactation. However, the T _r varied between different regions of the body with hind flippers on average 2–6°C warmer than all other areas measured. There was no difference in mean T _r of male and female pups and pup body mass did not account for the variation in T _r during early or late lactation. Throughout the day there was an increase in the T _r of pups and this explained 20–28% of the variation in T _r depending on stage of lactation. There was no difference in the mean T _r of pups between pupping sites or associated with different substrate types. Wind speed and substrate temperature had no effect on the T _r of pups. However, solar radiation, air temperature and relative humidity accounted for 48% of the variation in mean T _r of pups during early lactation. During late lactation air temperature and solar radiation alone accounted for 43% of the variation in T _r. These results indicate that environmental conditions explain only some of the variation in T _r of grey seal pups in natural conditions. Differences in T _r however indicate that the cost of thermoregulation for pups will vary throughout lactation. Further studies examining intrinsic factors such as blubber thickness and activity levels are necessary before developing reliable biophysical models for grey seals.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.