European checkerspots (Melitaeini: Lepidoptera,Nymphalidae) are not aposematic – the point of view of great tits (Parus major)

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Double trouble—Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments

The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant “ripple effect” on subsequent studies.     

DNA barcoding of South Africa’s ornamental freshwater fish – are the names reliable?

Trade in freshwater ornamental fish in South Africa is currently regulated by a ‘blacklist’ to prevent potentially invasive taxa from establishing in the country. Because its effective implementation requires accurate identification, the aim of the present study was to test whether DNA barcoding is a useful tool to identify freshwater fishes in the South African pet trade. A total of 351 aquarium fish specimens, representing 185 traded taxa, were sequenced for the mitochondrial COI barcoding marker in 2011 and 2012. Lake Malawi cichlids were treated as a single group due to a lack of resolution in their COI marker, resulting in a data set of 137 successfully sequenced taxa. The Barcode Of Life Database (BOLD) and GenBank were used for taxonomic assignment comparisons. The genetic identification matched the scientific name inferred from the trade name for 60 taxa (43.8%) using BOLD, and for 67 taxa (48.9%) using GenBank. A genetic ID could not be assigned in 47 (34.3%) cases using BOLD and in 37 cases (27%) using GenBank. Whereas DNA barcoding can be a useful tool to help identify imported freshwater fishes, it requires further development of publicly available databases to become a reliable means of identification.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Effects of citronella oil (Cymbopogon winterianus Jowitt ex Bor) on Spodoptera frugiperda (J. E. Smith) midgut and fat body

The armyworm, Spodoptera frugiperda, is the principal pest of corn in Brazil. Control is achieved primarily by synthetic insecticides, which cause problems for the agro-ecosystem. Alternative methods of control are under investigation and citronella (Cymbopogon winterianus) essential oil appears to be a promising agent. We investigated the effects of citronella oil using histological, histochemical and immunohistochemical methods. The midgut of larvae treated with citronella exhibited altered epithelium including cytoplasmic protrusions, columnar cell extrusion, pyknotic nuclei, and increased periodic acid-Schiff positive granules. Regenerative cells in the epithelium of the midgut increased in number, which facilitated subsequent regeneration of this tissue. After exposure to citronella, trophocytes, the principal cell type of the fat body, possessed enlarged vacuoles and mitotic bodies, and contained reduced amounts of glycogen, lipid, and protein. Citronella oil caused morphological changes of the midgut and reduction of stored resources in the fat body, which may adversely affect insect reproduction and survival.     

Structure and Function of the Peanut Panallergen Ara h 8

The incidence of peanut allergy continues to rise in the United States and Europe. Whereas exposure to the major allergens Ara h 1, 2, 3, and 6 can cause fatal anaphylaxis, exposure to the minor allergens usually does not. Ara h 8 is a minor allergen. Importantly, it is the minor food allergens that are thought to be responsible for oral allergy syndrome (OAS), in which sensitization to airborne allergens causes a Type 2 allergic reaction to ingested foods. Furthermore, it is believed that similar protein structure rather than a similar linear sequence is the cause of OAS. Bet v 1 from birch pollen is a common sensitizing agent, and OAS results when patients consume certain fruits, vegetables, tree nuts, and peanuts. Here, we report the three-dimensional structure of Ara h 8, a Bet v 1 homolog. The overall fold is very similar to that of Bet v 1, Api g 1 (celery), Gly m 4 (soy), and Pru av 1 (cherry). Ara h 8 binds the isoflavones quercetin and apigenin as well as resveratrol avidly.     

Sensilla coeloconica ringed by microtrichia in host‐seeking biting midges

The distribution and morphology of antennal sensilla coeloconica in parasitic and predaceous biting midges were studied in females of Forcipomyia (feeding on the blood of frogs), Atrichopogon (feeding on haemolymph), Austroconops, Culicoides (feeding on the blood of birds and mammals) and Brachypogon (feeding on haemolymph and dissolved tissues of insects) (all: Diptera: Ceratopogonidae). A Lower Cretaceous female of Archiculicoides (Diptera: Ceratopogonidae) from Lebanese amber, which fed on the blood of unknown vertebrates, was also examined. In sensilla coeloconica ringed by microtrichia, the peg is grooved longitudinally and protrudes distinctly from the pit. We suggest that the microtrichia encircling the protruding peg form a structure resembling a picket fence in order to maintain a higher level of humidity, which facilitates the capture and transport of odour molecules through the channels in the peg wall. Sensilla coeloconica ringed by microtrichia function as very effective chemoreceptors in host‐ and prey‐seeking activity. During the evolution of Ceratopogonidae, sensilla coeloconica with a fence of microtrichia have evolved twice in groups feeding on the blood of vertebrates (i.e. in the basal lineage: Lower Cretaceous or earlier) and in the subgenus Lasiohelea of Forcipomyia (Palaeogene). Sensilla coeloconica ringed by microtrichia are described for the first time in the relict genus Austroconops.     

华西银腊梅挥发油化学成分的研究

用水蒸气蒸馏法提取华西银腊梅挥发油,并用气相色谱-质谱(GC-MS)联用技术对其挥发油的化学成分进行分析,结果共鉴定了其中的39种成分,所鉴定成分含量约占总检出量的87.83%。其化学成分主要为(Z,Z)-9,12-十八碳二烯酸甲酯(9.00%),壬醛(5.83%),二十一烷(5.69%),二十烷(5.08%),辛炔酸(4.50%),2,6,10,15-四甲基十七烷(3.93%),(Z)-6-十八烯酸甲酯(3.65%),3,8-二甲基十一烷(3.52%),1-十六碳炔(3.31%),肉豆蔻酸(2.86%),月桂醛(2.81%),壬酸(2.23%),5,6,7,7α-四氢-4,4,7α三甲基-2(4H)-苯并呋喃酮(2.18%)等。     

Multiplex panels of polymorphic microsatellite loci for two cryptic bat species of the genus Pipistrellus,developed by cross‐species amplification within the family Vespertilionidae

The paper presents multiplex panels of polymorphic microsatellites for two closely related cryptic species Pipistrellus pipistrellus and Pipistrellus pygmaeus. We tested the cross‐species amplification of 34 microsatellite loci, originally developed for five vespertilionid bat species. Ten and nine polymorphic loci in P. pipistrellus (mean number of alleles per locus = 10.5) and P. pygmaeus (8.1), respectively, in three multiplex polymerase chain reactions per species were amplified. All loci can be analysed in a single fragment analysis and can be used as markers to the study of evolution and the ecology of structured populations of socially living bats.