A method for non-invasive genotyping of APCmin/+ mice using fecal samples

The APC^min/+ mouse is commonly used in cancer research and is just one of many genetically altered models that is currently being developed. With high numbers of breeding programs, it is important to have a simple method that can be used to genotype the mice non-invasively. Here we report a reproducible method for genotyping mice with DNA extracted from fecal samples. Comparison of fecal results with those obtained from intestinal tissue DNA and clinical outcome (presence/absence of tumors) showed this technique to have 100% accuracy. This non-invasive method of genotyping may be applied to other transgenic mouse models.     

Genetic characterization of Escherichia coli populations from host sources of fecal pollution by using DNA fingerprinting

Escherichia coli isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting. The genetic relationship of strains within each host group was assessed as was the relationship of strains among different host groups. Multiple isolates from a single host animal (gull, human, or dog) were found to be identical; however, in some of the animals, additional strains occurred at a lower frequency. REP PCR fingerprint patterns of isolates from sewage (n = 180), gulls (n = 133), and dairy cattle (n = 121) were diverse; within a host group, pairwise comparison similarity indices ranged from 98% to as low as 15%. A composite dendrogram of E. coli fingerprint patterns did not cluster the isolates into distinct host groups but rather produced numerous subclusters (approximately >80% similarity scores calculated with the cosine coefficient) that were nearly exclusive for a host group. Approximately 65% of the isolates analyzed were arranged into host-specific groups. Comparable results were obtained by using enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis (PFGE), where PFGE gave a higher differentiation of closely related strains than both PCR techniques. These results demonstrate that environmental studies with genetic comparisons to detect sources of E. coli contamination will require extensive isolation of strains to encompass E. coli strain diversity found in host sources of contamination. These findings will assist in the development of approaches to determine sources of fecal pollution, an effort important for protecting water resources and public health.     

Non-invasive genotyping of transgenic animals using fecal DNA

For genotyping of transgenic animals, many IACUC guidelines recommend the use of fecal DNA when possible because this approach is non-invasive. Existing methods for extracting fecal DNA may be costly or involve the use of toxic organic solvents. Furthermore, feces contain an abundance of PCR inhibitors that may hinder DNA amplification when they are co-purified with fecal DNA. Here the authors describe a cost-effective, non-toxic method for genotyping transgenic animals by using the reagent AquaStool to extract fecal DNA and remove PCR inhibitors. Genotyping results obtained from fecal DNA samples extracted using AquaStool were reliably accurate when compared with results obtained from tail DNA samples. Because it is non-invasive, the authors believe that use of this method for genotyping transgenic animals using fecal DNA samples may improve animal welfare.     

Rapid, one-step DNA extraction for insect pest identification by using DNA barcodes

Early detection of economically important insects is critical to preventing their establishment as serious pests. To accomplish this, tools for rapid and accurate species identification are needed. DNA barcoding, using short DNA sequences as species "genetic identification tags," has already shown large potential as a tool for rapid and accurate detection of economically important insects. DNA extraction is the critical first step in generating DNA barcodes and can be a rate-limiting step in very large barcoding studies. Consequently, a DNA extraction method that is rapid, easy to use, cost-effective, robust enough to cope with range of qualities and quantities of tissue, and can be adapted to robotic systems will provide the best method for high-throughput production of DNA barcodes. We tested the performance of a new commercial kit (prepGEM), which uses a novel, streamlined approach to DNA extraction, and we compared it with two other commercial kits (ChargeSwitch and Aquapure), which differ in their method of DNA extraction. We compared performance of these kits by measuring percentage of polymerase chain reaction (PCR) success and mean PCR product yield across a variety of arthropod taxa, whichincluded freshly collected, ethanol-preserved, and dried specimens of different ages. ChargeSwitch and prepGEM performed equally well, but they outperformed Aquapure. prepGEM was much faster, easier to use, and cheaper than ChargeSwitch, but ChargeSwitch performed slightly better for older (> 5-yr-old) dried insect specimens. Overall, prepGEM may provide a highly streamlined method of DNA extraction for fresh, ethanol-preserved, and young, dried specimens, especially when adapted for high-throughput, robotic systems.     

Estimating mountain goat abundance using DNA from fecal pellets

Non-invasive collection of tissue samples to obtain DNA for microsatellite genotyping required to estimate population size has been used for many wildlife species but rarely for ungulates. We estimated mountain goat (Oreamnos americanus) population size on a mountain complex in southwestern British Columbia by identification of individuals using DNA obtained from fecal pellet and hair samples collected during 3 sampling sessions. We identified 55 individuals from 170 samples that were successfully genotyped, and estimated a population of 77 mountain goats (SE = 7.4). Mean capture probability was 0.38 (SE = 0.037) per session. Our technique provides one of the first statistically rigorous estimates of abundance of an ungulate species using DNA derived primarily from fecal pellets. Our technique enables managers to obtain minimum counts or population estimates of ungulates in areas of low sightability that can be used for conservation and management. © 2011 The Wildlife Society.     

Validation of swabs as a non-destructive and relatively non-invasive DNA sampling method in fish

Non-destructive methods of collecting DNA from small fish species can be problematic, as fin clips can potentially affect behaviour or survivorship in the wild. Swabbing body mucus may provide a less invasive method of DNA collection. However, risk of contamination from other individuals in high density groups could give erroneous genotyping results. We compared multilocus microsatellite genotypes from the same individuals when collected at low and high density and compared this with fin clips. We found no differences between these categories, with a genotyping error rate of 0.42%, validating the use of body mucus swabbing for DNA collection in fish.     

Incorporation of antifreeze proteins into zebrafish embryos by a non-invasive method

The cryopreservation of fish embryos is a challenge because of their structure, with multiple compartments and permeability barriers, and their high chilling sensitivity. Vitrification at advanced developmental stages is considered to be the more promising option. Nevertheless, all reported attempts have failed. Previous studies demonstrated a better ability for freezing in species that naturally express antifreeze proteins (AFPs). These proteins have been delivered into other fish embryos using time-consuming techniques like microinjection. In the present study, the introduction of FITC labelled AFPs was assayed in zebrafish embryos at early developmental stages (from 2-cell to high blastula stage), before the formation of the yolk syncytial layer, by an easy and non-invasive method and evaluated by fluorescence and confocal microscopy. Incubation with AFPs at 128-cell or high blastula stage provides incorporation of the protein in 50–90% of embryos without affecting hatching. Incubation in media containing protein is a simple, harmless and effective method which makes it possible to treat several embryos at the same time. AFPs remain located in derivatives from marginal blastomeres: the yolk syncytial layer, the most cryosensitive and impermeable barrier, and different digestive organs. Our findings demonstrate that delivery of AFP type I and AFP type III into zebrafish embryos by incubation in media containing protein is a simple and harmless method that may improve cryoprotection of the cellular compartment.     

Detecting predation and scavenging by DNA gut-content analysis: a case study using a soil insect predator-prey system

White grubs (larvae of Coleoptera: Scarabaeidae) are abundant in below-ground systems and can cause considerable damage to a wide variety of crops by feeding on roots. White grub populations may be controlled by natural enemies, but the predator guild of the European species is barely known. Trophic interactions within soil food webs are difficult to study with conventional methods. Therefore, a polymerase chain reaction (PCR)-based approach was developed to investigate, for the first time, a soil insect predator-prey system. Can, however, highly sensitive detection methods identify carrion prey in predators, as has been shown for fresh prey? Fresh Melolontha melolontha (L.) larvae and 1- to 9-day-old carcasses were presented to Poecilus versicolor Sturm larvae. Mitochondrial cytochrome oxidase subunit I fragments of the prey, 175, 327 and 387 bp long, were detectable in 50% of the predators 32 h after feeding. Detectability decreased to 18% when a 585 bp sequence was amplified. Meal size and digestion capacity of individual predators had no influence on prey detection. Although prey consumption was negatively correlated with cadaver age, carrion prey could be detected by PCR as efficiently as fresh prey irrespective of carrion age. This is the first proof that PCR-based techniques are highly efficient and sensitive, both in fresh and carrion prey detection. Thus, if active predation has to be distinguished from scavenging, then additional approaches are needed to interpret the picture of prey choice derived by highly sensitive detection methods.     

Modulation of gastric motility by brain-gut peptides using a novel non-invasive miniaturized pressure transducer method in anesthetized rodents

Acute in vivo measurements are often the initial, most practicable approach used to investigate the effects of novel compounds or genetic manipulations on the regulation of gastric motility. Such acute methods typically involve either surgical implantation of devices or require intragastric perfusion of solutions, which can substantially alter gastric activity and may require extended periods of time to allow stabilization or recovery of the preparation. We validated a simple, non-invasive novel method to measure acutely gastric contractility, using a solid-state catheter pressure transducer inserted orally into the gastric corpus, in fasted, anesthetized rats or mice. The area under the curve of the phasic component (pAUC) of intragastric pressure (IGP) was obtained from continuous manometric recordings of basal activity and in responses to central or peripheral activation of cholinergic pathways, or to abdominal surgery. In rats, intravenous ghrelin or intracisternal injection of the thyrotropin-releasing hormone agonist, RX-77368, significantly increased pAUC while coeliotomy and cacal palpation induced a rapid onset inhibition of phasic activity lasting for the 1-h recording period. In mice, RX-77368 injected into the lateral brain ventricle induced high-amplitude contractions, and carbachol injected intraperitoneally increased pAUC significantly, while coeliotomy and cecal palpation inhibited baseline contractile activity. In wild-type mice, cold exposure (15 min) increased gastric phasic activity and tone, while there was no gastric response in corticotropin releasing factor (CRF)-overexpressing mice, a model of chronic stress. Thus, the novel solid-state manometric approach provides a simple, reliable means for acute pharmacological studies of gastric motility effects in rodents. Using this method we established in mice that the gastric motility response to central vagal activation is impaired under chronic expression of CRF.     

Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 x 10(-5) to 2.8 x 10(-7) g (dry weight) of feces/liter and 6.8 x 10(-7) g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.     

Estimating abundance of Sitka black-tailed deer using DNA from fecal pellets

Densely vegetated environments have hindered collection of basic population parameters on forest-dwelling ungulates. Our objective was to develop a mark–recapture technique that used DNA from fecal pellets to overcome constraints associated with estimating abundance of ungulates in landscapes where direct observation is difficult. We tested our technique on Sitka black-tailed deer (Odocoileus hemionus sitkensis) in the temperate coastal rainforest of Southeast Alaska. During 2006–2008, we sampled fecal pellets of deer along trail transects in 3 intensively logged watersheds on Prince of Wales Island, Alaska. We extracted DNA from the surface of fecal pellets and used microsatellite markers to identify individual deer. With genotypes of individual deer, we estimated abundance of deer with moderate precision (±20%) using mark–recapture models. Combining all study sites, we identified a 30% (SE = 5.1%) decline in abundance during our 3-year study, which we attributed to 3 consecutive severe winters. We determined that deer densities in managed land logged >30 years ago (7 deer/km², SE = 1.3) supported fewer deer compared to both managed land logged <30 years ago (10 deer/km², SE = 1.5) and unmanaged land (12 deer/km², SE = 1.4). Our study provides the first estimates of abundance (based on individually identified deer) for Sitka black-tailed deer and the first estimates of abundance of an unenclosed ungulate population using DNA from fecal pellets. Our tool enables managers to accurately and precisely estimate the abundance of deer in densely vegetated habitats using a non-invasive approach. © 2011 The Wildlife Society.     

Calibrating phylogenetic species formation in a threatened insect using DNA from historical specimens

Museum specimens from the late 19th and early 20th centuries were surveyed for the single nucleotide polymorphism identified previously and used to diagnose populations of the federally threatened Northeastern Beach Tiger Beetle Cicindela d. dorsalis (Coleoptera: Carabidae). Widespread polymorphism was revealed throughout the historical range of this species, suggesting a relatively recent anthropogenic character fixation event associated with the extinction and fragmentation of populations. Implications for the phylogenetic species criterion and for the reintroduction of individuals to formerly occupied sites are discussed.     

Reconstruction of kinship by fecal DNA analysis of orangutans

Genetic analysis is a useful tool for assigning biological relationships. Thus, it will improve genetic management of wild animal populations and breeding colonies. Kinship analysis will give new insights into the behavior, sociobiology and genetic management of orangutans. In this study, chromosomal DNA from orangutan (Pongo pygmaeus ssp.) was extracted from excrements. Feces samples were screened for up to nine microsatellite markers from related zoo populations of orangutans (Pongo pygmaeus ssp.) kept at the Zoological Garden Berlin and the Zoological Garden Heidelberg, Germany. Family structures are documented in the "International Studybook of the Orangutan" (Perkins 1995) and the "Europ?isches Erhaltungszucht Programm 1998" (Becker 1998). To examine whether human short tandem repeat loci (STR) are suitable for the reconstruction of kinship in orangutans, nine STRs, commonly used in forensic studies and the amelogenin system, were amplified in a multiplex-PCR approach (AmpFlSTR Profiler Plus). We were able to show that five of the nine human autosomal STRs in question amplified successfully in orangutans. Thus, we could reconstruct kinship structures of the Berlin and Heidelberg populations.     

Monitoring of hitchhiker insect pests collected on foreign vessels entering Korea using by DNA barcodes

We monitored hitchhiker insect pests on vessels arriving to Korea from foreign countries. A survey of 416 foreign vessels was conducted from June 1, 2018 to October 29, 2019. A total of 1,141 hitchhiker insects were collected by interception or by hand. Individuals were identified to the species level whenever possible using DNA barcoding and morphological re-examination. Of the 1,141 hitchhikers, a total of 439 species from 81 families and 12 orders, including 54 species (93 individuals) not-distributed in Korea, were identified. Among the 93 individuals from the 54 species not distributed in Korea, 78 individuals were collected alive. In particular, three regulated species in Korea, Erthesina fullo (Pentatomidae, Hemiptera) (hitchhiking route: China > Yeosu, Korea), Tessaratoma papillosa (Tessaratomidae, Hemiptera) (China > Yeosu, Korea), Noctua pronuba (Noctuidae, Lepidoptera) (Singapore > Pohang, Korea) were detected, nine species, Dicranocephalus wallichii (Scarabaeidae, Coleoptera), Palpita quadristigmalis (Crambidae, Lepidoptera), Lymantria xylina (Erebidae, Lepidoptera), Odontopera aurata, Semiothisa cinerearia (Geometridae, Lepidoptera), Mythimna pallidicosta (Noctuidae, Lepidoptera), Euhampsonia serratifera (Notodontidae, Lepidoptera), Arippara disticha (Pyralidae, Lepidoptera), and Psilogramma lukhtanovi (Sphingidae, Lepidoptera) were discovered as multiple detection over years or months, and three species, Lemyra rhodophilodes (Erebidae, Lepidoptera), Malacosoma dentata (Lasiocampidae, Lepidoptera), and Chondracris rosea (Acrididae, Orthoptera) were multiple detected from single vessel. Therefore, we suggest that hitchhiker monitoring on vessels travelling along navigated routes should be conducted in addition to performing a risk assessment of hitchhiker insect pest species not distributed in Korea that includes regulated species.     

Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed. Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions. Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation. These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients.