Fluorescent Amplified Fragment Length Polymorphism Research Articles

Fluorescent amplified fragment length polymorphism (fAFLP) analysis of Listeria monocytogenes.

Fluorescent amplified fragment length polymorphism (fAFLP) is based on the selective PCR amplification of restriction fragments from a digest of total genomic DNA. Genomic DNA extracted from a purified bacterial isolate is completely digested with two endonucleases generating fragments which are ligated to specific double-stranded adaptors. The ligated fragments are then amplified by PCR using fluorescently labelled primers. Fluorescent amplified fragments are separated by size on an automated sequencer with a size standard. fAFLP is a rapid, highly reproducible technique which can be used to discriminate and subtype Listeria monocytogenes strains.

Construction of a New Molecular Barcode for Discriminating Plants and Animals with a Close Genetic Relationship

DNA barcodes have been proposed as a shortcut to provide species identification and as a way to accelerate the discovery of new species. A number of candidate gene regions have been suggested as possible barcodes for animals and plants, but for the identification of recently diverged species and/or varieties with only a few genetic differences it has been reported to be problematic in some cases. This study selected widely cultivated cruciferous vegetables as the primary samples, after failure of discrimination of each species using current DNA barcodes, we performed the fluorescent amplified fragment length polymorphism (F-AFLP) and successfully discriminated each species, subspecies, variety and their cultivar in 74 samples. Then the non-qualitative results obtained from F-AFLP were transformed into two-dimensional barcodes image file of each cultivar via the PDF417 software. This method was also successfully applied to the discrimination of 17 Chinese indigenous pig breeds. The barcode we constructed which greatly reduces the information storage space is genotypes-specific, and can be conveniently decoded into the original data and thereby be conveniently shared and referred to. We believe that it is possible to construct a new data sharing molecular barcode system that could discriminate the subspecies, varieties, cultivars and even individuals with close genetic relationships.

Streptococcus rubneri sp. nov., isolated from the human throat

The novel, Gram-stain-positive, ovoid, lactic acid bacterial isolates LMG 27205, LMG 27206, LMG 27207(T) and MRI-F 18 were obtained from throat samples of healthy humans. 16S rRNA gene sequence analyses indicated that these isolates belong to the genus Streptococcus, specifically the Streptococcus mitis group, with Streptococcus australis and Streptococcus mitis as the nearest neighbours (99.45 and 98.56 % 16S rRNA gene sequence similarity to the respective type strains). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE), DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA and atpA and physiological and biochemical tests revealed that these bacteria formed a taxon well separated from its nearest neighbours and other species of the genus Streptococcus with validly published names and, therefore, represent a novel species, for which the name Streptococcus rubneri sp. nov. is proposed, with LMG 27207(T) ( = DSM 26920(T)) as the type strain.

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping

BackgroundListeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES’s Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL) network. Since 2008, the United Kingdom (UK)-NRL for L. monocytogenes at the Health Protection Agency (HPA), London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP) for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes.ResultsA panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively.ConclusionsThe discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human listeriosis and tracking the source of contamination in food processing facilities in real time.

Insertion site mapping for repeated elements in Mycobacterium tuberculosis

Insertion elements not only act as genetic markers for differentiation of bacteria but their movement in bacterial genomes likely plays an essential role in changing the physical and biochemical traits of the organisms when adapting to new environments. Genomic Insertion Site mapping of transposable elements could shed light on the putative altered function of adjacent genes. In the era of whole genome sequencing where repeat elements are difficult to sequence with short read technologies and in the absence of high throughput technologies especially in poorer resource settings, an alternative approach to their characterisation is needed. A rapid and simple method of insertion site mapping that uses Insertion Sequence 6110 (IS6110) fluorescent amplified fragment length polymorphism (FAFLP) PCR as a foundation and then uses additional selective bases to reduce the number of fragments generated was developed. This was applied to Mycobacterium tuberculosis H37Rv sequenced strain to compare the experimental data with the in silico results. This was successfully achieved for all but two of the sixteen fragments generated by FAFLP and demonstrated that, by using this technique, insertion sites can be mapped onto the genomes of M. tuberculosis.

Pediococcus lolii DSM 19927T and JCM 15055T are strains of Pediococcus acidilactici

Strain NGRI 0510Q(T), isolated from ryegrass silage, was recently classified as a representative of a novel Pediococcus species, Pediococcus lolii Doi et al. 2009. It was deposited in the DSMZ and JCM culture collections as DSM 19927(T) and JCM 15055(T), respectively. A polyphasic taxonomic study, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, pheS and 16S rRNA gene sequence analysis, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization, was used to prove that both subcultures of the type, and only, strain of this species are strains of Pediococcus acidilactici.

Mapping the genetic diversity within major clonal complexes of meticillin-resistant Staphylococcus aureus utilizing genome-wide fluorescent amplified fragment length polymorphism markers

The genetic diversity between major meticillin-resistant Staphylococcus aureus (MRSA) lineages was probed using fluorescent amplified fragment length polymorphism (FAFLP) as a random genome sampling tool. Genomic DNA was digested with endonucleases BglII and Csp6I and a subset of the restricted fragments were amplified using the primer pair BglII+A and Csp6I+0. Sixty-seven FAFLP profiles consisting of 46-68 amplified fragments ranging in size from 50 to 600 bp were exhibited amongst the 71 isolates analysed. Cluster analysis of FAFLP data revealed concordance with spa typing and MLST clonal complexes (CC), with isolates of each CC grouping in the same FAFLP cluster. Furthermore, FAFLP could differentiate subtypes within the homogeneous CC22 isolates and also between MLST sequence types 8 and 239. The discriminatory power of FAFLP was 0.998 compared to values of 0.975 and 0.909 for spa typing and MLST, respectively. Thus, FAFLP analysis proved to be a rapid, reproducible and high-resolution tool that displayed the microheterogeneity within MRSA lineages. Using FAFLP data, lineage-specific fragments were identified and sequenced; these encoded toxins, antibiotic resistance determinants and bacteriophage resistance factors. Lineage-specific sequence variations were observed, which may provide insights into the evolution and fitness of successful lineages. This will also aid in the development of rapid and high-throughput diagnostic PCR-based assays for the identification of MRSA lineages in resource-poor settings.

Limited divergence among populations of rice striped stem borer in southeast China caused by gene flow: Implications for resistance management

Abstract Rice striped stem borer (RSSB, Chilo suppressalis) is a serious lepidopteron pest occurring in rice ecosystems of Asia and Europe. Genetically modified (GM) insect‐resistant Bt rice has been developed to deter lepidopteron pests including RSSB. The concern of resistance evolution to the Bt toxin by the pests under commercial cultivation of GM Bt rice and the need of effective management of the resistance encourage the studies of genetic variation and divergence, as well as gene flow of RSSB populations. We analyzed 13 RSSB populations fed on water‐oats or rice plants, respectively, from southeast China applying the fluorescent amplified fragment length polymorphism fingerprints. A generally moderate level of genetic variation was detected in the populations, as estimated by Nei's genetic diversity (0.27) and Shannon's index (0.42). The FST and amova values indicated a low level (∼12%) of genetic divergence among the RSSB populations. A relatively frequent gene flow (an average Nm = 2.62) was detected among the 12 RSSB populations, which may explain the limited genetic divergence among the rice‐feeding populations. This explanation gains support by the assignment test of the corresponding populations, suggesting that a considerable proportion of individuals was contributed from non‐native populations. Our results revealed that the moderate level of genetic diversity combined with relatively frequent gene flow among RSSB populations across a large geographical range may slow down the resistance evolution of the RSSB populations, given that a proper measure of resistance management is taken.

DETECTION AND IDENTIFICATION METHODS AND NEW TESTS AS DEVELOPED AND USED IN THE FRAMEWORK OF COST 873 FOR BACTERIA PATHOGENIC TO STONE FRUITS AND NUTS

Xanthomonas arboricola pv. juglandis (Pierce) Vauterin et al. (1995) is the causal agent of walnut bacterial blight, the major nut and foliage disease of Persian (English) walnut (Juglans regia). The disease limits walnut production in most regions of the world. All new walnut tissues are susceptible to infection and necrosis can occur in catkins, female flowers, leaves, fruit and green shoots (Miller and Bollen, 1946). Over time, the bacterium has been classified into different genera under different names, i.e. Pseudomonas juglandis Pierce (1901); Bacterium juglandis (Pierce) Smith (1905); Phytomonas juglandis (Pierce) Bergey et al. (1930); Xanthomonas juglandis (Pierce) Dowson (1939) and Xanthomonas campestris pv. juglandis (Pierce) Dye (1978), which is officially accepted in the International standards for naming pathovars of phytopathogenic bacteria (Dye et al., 1980). Recently, however, it was re-classified by Vauterin et al. (1995) as Xanthomonas arboricola pv. juglandis (Xaj). Xaj is the causal agent of two new emerging diseases of Persian (English) walnut (J. regia) known as vertical oozing canker (VOC) (Hajri et al., 2010), and apical necrosis (Moragrega and Ozaktan, 2010; Moragrega et al., 2011). According to molecular studies based on fluorescent amplified fragment length polymorphism (FAFLP), in France VOC is caused by a distinct genetic lineage of the bacterium which is also able to induce blight symptoms on leaves and fruits (Hajri et al., 2010). Bacterial strains isolated from apical necrosis lesions in Spain are also able to infect walnut leaves, causing typical blight lesions (Moragrega et al., 2011).

Use of AFLP to study the genetic variability of Leishmania sp

AFLP is a very efficient technique for rapid detection of genetic variability, particularly useful on organisms without sequenced genomes. The objective of this study was to assess the amplified fragment length polymorphisms (AFLP) for testing genetic diversity in Leishmania sp. Leishmania species used for this study were representative of the Viannia and Leishmania subgenus. Fluorescent AFLP kits (Applied Biosystems, USA) were used for the whole procedure. Detection and sizing of peaks were conducted with GeneMarker v. 1.91 software (Softgenetics, USA). Then error rate was estimated for every possible pair of replicates using ten replicates from a single L. panamensis promastigotes culture. We have found that this technique is able to generate high numbers of peaks when low selective EcoRI and MseI primers were used (+0, +1, +2 series). Additionally, we have found that up to 86,9% of the primer combinations are polymorphic. Several fragments were apparently specific for L panamensis, L. (Viannia) and L. (Leishmania). The error rates estimated from every possible pair of replicates ranged from 0% to 6%. Cluster analysis results are in agreement with the current known taxonomy of Leishmania species. In conclusion, AFLP is a molecular fingerprinting technique showing promising potential to explore genetic diversity at Leishmania sp., both at intraspecific and interspecific levels. Its application will allow researchers to look deeper into new polymorphisms and possibly correlate them to ecoepidemiologically relevant traits and other important aspects of the biology of the parasite.

Genetic analysis of a garlic (Allium sativum L.) germplasm collection from Argentina

Although garlic spreads asexually, it shows a wide diversity for physiologic, morphologic and agronomic characters, due to the accumulation of mutations. Our objective was to examine the genetic diversity of a collection of 43 national and foreign introductions using fluorescent Amplified Fragment Length Polymorphism (fAFLP) markers for characterizing previously phenotypical. 351 bands were obtained, of those 251 were polymorphic. The obtained genotypes were analyzed by means of the program Structure v2.2 according to a mixture model of frequencies correlated by allelic population. Twenty simulations of 55,000 steps defaulting from two to eight different genetic populations were carried out. The solutions with more likelihood were obtained by conforming five groups of clones. Most of the individuals showed compound genotypes for portions of two or more populations and duplications were identified. If the phenotypical classifications are correct, the phenotype can respond to different genetic origins, including individuals genetically atypical in a uniformly phenotypical population. Some clones were isolated after multiple selections on populations of garlic, what is not surprising, is that some are more genetically related than others. The genetic relationships found in the collection of garlic belong together with the classification of the ecophysiological groups, although not in a definitive way, additional analysis being necessary. The conservation of the genetic diversity is of vital importance because it guarantees a broader background with which to practice artificial selection assuring the success of future programs of improvement.

AFLP analysis and zebra disease resistance identification of 40 sisal genotypes in China

Sisal is the most important fiber crop in tropical and subtropical areas in China and the world. Zebra disease is a serious threat to the main cultivar Agave hybrid No.11648 (H.11648) worldwide. To select germplasm materials with zebra disease resistance for breeding, the fluorescent amplified fragment length polymorphism (AFLP) technique was used to make a cluster analysis of the genetic relationships of 40 sisal genotypes grown in China, and Phytophthora nicotianae was used to inoculate the 40 genotypes to identify their resistance to zebra disease. As a result, the similarity coefficient among 40 sisal genotypes was found to be 0.44-0.83 and the 40 genotypes show different levels of disease resistance. According to the AFLP analysis, the disease resistance and chromosomal ploidy, it can be reasoned that, A. attenuata var. marginata, Dong 109, Nan ya 1 and A. attenuata are suitable for hybridization with H.11648 to breed a new disease-resistant variety.

Genetic characterization of pomegranate (Punica granatum L.) genotypes by AFLP markers.

The Coruh Valley, located in Northeastern Turkey, is one of the most important centers of diversity in pomegranate in Turkey. In this study, we attempted to characterize 19 promising pomegranate genotypes originating from the Coruh Valley in using fluorescent dye AFLP markers and capillary electrophoresis. Four AFLP primer combinations were used, generating a total of 297 fragments, 213 of which were polymorphic (73.0%). Resolving powers of the AFLP primers ranged from 0.700 to 1.018, with a total of 3.440, while polymorphism information contents ranged from 0.707 to 0.837 with an average of 0.764. UPGMA clustering of the genotypes showed two major groups. Most of the fruit characteristics of the genotypes within the same group were variable. Therefore, the results showed that molecular characterization is necessary to get reliable relationships among pomegranate genotypes and AFLP markers can be used effectively in pomegranate.

Molecular Approaches in the Detection and Characterization of Leptospira

Conventional diagnosis of leptospirosis and characterization of Leptospira spp. relies mainly on serology. A major drawback of serology in diagnosis is that it needs sufficient levels of anti-Leptospira antibodies, thus jeopardizing confirmation in the early acute phase of disease. The cross agglutinin absorption test (CAAT) that determines Leptospira serovars is a technically demanding and laborious method and therefore is only performed at a few laboratories. Novel molecular diagnostic tests mainly rely on the polymerase chain reaction (PCR). PCR perfectly complements serological testing, since it is especially sensitive in the first 5 days after onset of symptoms. Real time PCR is rapid and has been validated for their high clinical accuracy. The introduction of molecular techniques for defining Leptospira species revolutionized the categorization of strains in this genus, as species and serogroups appeared to show little correlation. The reference test in molecular speciation is based on determining DNA homology. This approach is tedious and user-unfriendly and therefore is increasingly replaced by other techniques. To date, a wealth of molecular typing methods is available. Most attractive are those techniques that provide direct digital and electronically portable data. Such techniques comprise fluorescent amplified fragment length polymorphism (FAFLP), array typing, multilocus variable number of tandem repeats analysis (MLVA) and sequence-based phylogeny, and to some extent pulsed field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) is the most robust method for determining Leptospira strain diversity and in the future will probably only be surpassed by phylogeny on whole genome sequences.

Phenotypic and Genotypic Characterization of Coagulase Negative Staphylococci (CoNS) Other thanStaphylococcus epidermidisIsolated from Ocular Infections

The clinical significance of many species belonging to coagulase-negative staphylococci (CoNS) other than Staphylococcus epidermidis has been increasingly recognized. The present study attempted the characterization of non-S. epidermidis CoNS isolates of ocular origin. The isolates were characterized phenotypically by analytical profile index (API) and genotypically by fluorescent amplified fragment length polymorphism (FAFLP). In addition, the presence of genes that are likely to enhance virulence such as biofilm related genes (icaA and icaB) and methicillin resistance (mecA) were detected. API identified seven different species with an identification score varying from 44% to 99%. S. haemolyticus and S. xylosus species identified by API showed good API scores when compared with all other species identified. FAFLP generated 12 clusters from all the isolates and appeared to be more discriminatory. API identification results corresponded well with the FAFLP results only in six clusters. Nearly 40% of isolates showed the presence of mecA and icaAB genes. Identification results of these two methods corresponded only in 48.4% of the isolates suggesting that the battery of tests using API were not sufficient enough to identify all the species of CoNS. Therefore, it is sensible to rely on two or more identification methods particularly when API species identification has a lower score. FAFLP genotyping appears to be a reliable and rapid alternative identification method for CoNS that can be used in conjunction with any other phenotypic method.

Genetic Analysis of Walnut Cultivars in China Using Fluorescent Amplified Fragment Length Polymorphism

Informative DNA fingerprints from 50 walnut cultivars (Juglans regia) in China were generated using amplified fragment length polymorphism (AFLP) markers to reveal their genetic diversity and relationships. Nine primer combinations were selected from 64 EcoR I/Mse I primer combinations to amplify the accessions. An average of 132 polymorphic loci per primer set was detected from the nine primer combinations. The discrimination power of each polymorphic marker (estimated by the polymorphism information content) ranged from 0.00 to 0.37 with an average of 0.19. A moderate level of genetic diversity was observed among the 50 cultivars. Their expected heterozygosity varied from 0.38 to 0.50 (average, 0.44), and Dice's similarity coefficient ranged from 0.53 to 0.86 (average, 0.70). The cluster analysis conducted using the unweighted pair group method of arithmetic averages method showed that all of the cultivars fell into five groups at Dice's similarity coefficient of 0.68. According to the comprehensive analyses based on the specific loci, similarity coefficient, and clustering results, six cultivars (Liaoning 1, Zixin, Shanhe 4, Zha 343, Tulare, and Chandler) were considered important germplasms of walnut cultivars.

Distinct immunomodulatory properties of Lactobacillus paracasei strains

This study was performed to ascertain the immunomodulatory effect of Lactobacillus paracasei strains. These strains were also genetically characterized. The strains were genetically differentiated by using the fluorescent-amplified fragment length polymorphism technique, which led to the identification of several molecular markers unique to each strain. To determine the immunomodulatory properties, we evaluated the effect of strains on dendritic cell maturation, dextran uptake, ability to induce proliferation of allogenic T cells and cytokine secretion. The results indicated that all the strains stimulated phenotypic maturation of dendritic cells (DCs), but they acted differently on DCs in relation to the other tested properties; notably, a different effect on cytokine secretion was detected. The results of this study revealed different immunomodulatory properties of strains of the species Lact. paracasei. Strain IMPC 4.1 showed an interesting anti-inflammatory ability. Probiotic strains IMPC 2.1 and LMG P-17806 were characterized by a similar and intermediate ability to induce cytokine secretion in contrast to the very low ability of strain LMG 23554. Our results confirm that each single strain of a bacterial species appears to influence the immune system in a peculiar manner. The evaluation of the different types and/or levels of cytokines whose secretion is induced by each strain could be relevant to define its pro- or anti-inflammatory properties and its more appropriate clinical use.

Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials.

Fluorescent amplified fragment length polymorphism (AFLP) analysis of genetic diversity and relationship of Chinese Rosa rugosa germplasm resources

Rosa rugosa is an excellent ornamental plant with important ecological, economical and medicinal values in China. Polymorphism amplifications of the genomic DNA of 5 wild R. rugosa accessions and 25 cultivars that originated from China with fluorescent amplified fragment length polymorphism molecular markers were investigated in this study to evaluate their genetic diversity and relationships. A total of 1771 clearly identifiable bands (60 ~ 500 bp), including 1704 polymorphic bands, were obtained through amplification using 10 pairs of primers. The average percentage of polymorphic locus was 96.2%, which indicated the high genetic diversity of Chinese R. rugosa germplasm resources. Different quantities of specific bands were detected by 10 pairs of primers from 24 accessions, which could be used to identify the specific R. rugosa accession. There was high similarity between Chinese R. rugosa germplasm resources, as indicated by the similarity coefficient of 30 R. rugosa accessions between 0.4977 and 0.8410, with an average of 0.624. Clustering results suggested that wild accessions from different areas of China were remotely related with the ample genetic diversity. Wild accessions and cultivars also had remote genetic relationship. However, the main Chinese R. rugosa cultivars had high similarity, low genetic differences and a narrow genetic background. Key words : Rosa rugosa, genetic diversity and relationship, amplified fragment length polymorphism.

Loss of genetic diversity in the North American mayfly Ephemerella invaria associated with deforestation of headwater streams

Summary 1. Terrestrial dispersal by aquatic insects increases population connectivity in some stream species by allowing individuals to move outside the structure of the stream network. In addition, individual survival and reproductive success (as well as dispersal) are tightly linked to the quality of the terrestrial habitat. 2. In historically forested catchments, deforestation and altered land use have the potential to interfere with mayfly dispersal or mating behaviours by degrading the quality of the terrestrial matrix among headwater streams. We hypothesised that loss of tree cover in first-order catchments would be associated with an increase in population substructure and a decrease in genetic diversity of mayfly populations. 3. To test this hypothesis, we investigated spatial patterns of genetic variation in the common mayfly Ephemerella invaria across a gradient of deforestation in the central piedmont region of eastern United States. Intraspecific genetic diversity and population substructure were estimated from data obtained using fluorescent amplified fragment length polymorphism (AFLP) markers. 4. We found that mayfly populations had low population substructure within headwater stream networks and that genetic diversity was strongly negatively correlated with mean deforestation of the first-order catchments. The large-scale pattern of population substructure followed a pattern of isolation by distance (IBD) in which genetic differentiation increases with geographical distance, but assignment tests placed a few individuals into populations 300 km away from the collection site. 5. Our results show that loss of genetic diversity in this widespread aquatic insect species is co-occurring with deforestation of headwater streams. 6. Most arguments supporting protection of headwater streams in the United States have centred on the role of these streams as hydrological and biogeochemical conduits to downstream waters. Our work suggests that headwater stream land use, and specifically tree cover, may have a role in the maintenance of regional genetic diversity in some common aquatic insect species.