Development of a quantitative PCR method for screening ichthyoplankton samples for bigheaded carps

Monitoring ichthyoplankton is useful for identifying reproductive fronts and spawning locations of bigheaded carps (Hypophthalmichthys spp.). Unfortunately, sorting and identifying ichthyoplankton to monitor for bigheaded carp reproduction is time consuming and expensive. Traditional methods require frequent egg-larvae sampling, sorting of all samples to obtain presumptively identified bigheaded carp, and genetic validation of presumptively identified eggs. Quantitative PCR (qPCR) has the potential to streamline this process by identifying samples that likely do or do not contain a target species. Our objective was to develop a genetic screening tool using qPCR with the duplex assays SCTM4/5 and BHTM1/2 to prioritize samples that have a higher likelihood of containing bigheaded carp eggs or larvae. We used tandem ichthyoplankton samples collected for monitoring bigheaded carps in the Upper Mississippi, Illinois, and St. Croix rivers to evaluate the effectiveness of qPCR as a screening tool. Samples with \u3e 10,000 copies of DNA had 100% occurrence of bigheaded carp eggs or larvae in the traditionally sorted samples, whereas samples with \u3c 10 copies of DNA had 0% occurrence of ichthyoplankton from these invasive species. We used a logistic regression model to calculate the probability of finding bigheaded carp eggs or larvae based upon the number of DNA copies; 406 copies corresponded with a 50% probability of having bigheaded carp ichthyoplankton present in a sample. These data can be used to inform management actions (i.e., control, containment) for these invasive fishes, and this tool could be adapted for monitoring for reproduction of other aquatic invasive species

Validation of a Portable eDNA Detection Kit for Invasive Carps

Loop-mediated isothermal amplification (LAMP) is a rapid molecular detection technique that has been used as a diagnostic tool for detecting human and animal pathogens for over 20 years and is promising for detecting environmental DNA shed by invasive species. We designed a LAMP assay to detect the invasive carps, silver carp (Hypophthalmichthys molitrix), bighead carp (Hypophthalmichthys nobilis), black carp (Mylopharyngodon piceus), and grass carp (Ctenopharyngodon idella). To determine the sensitivity of the LAMP assay, we determined limit of detection (LOD) for each invasive carp species and compared with the performance of a grass carp quantitative PCR (qPCR) assay in LOD and in a mesocosm study. We used two grass carp densities, 3 juvenile grass carp in one mesocosm and 33 juvenile grass carp in the other. Prior to adding grass carp to the mesocosms, we added 68 kg of fathead minnows (Pimephales promelas) to each mesocosm to simulate farm ponds used for raising bait fish. We filtered 500 mL of water per sample to compare LAMP and qPCR analysis, and we collected 50 mL grab samples that were only analyzed using qPCR to gain additional data using a higher-throughput method to monitor environmental DNA (eDNA) levels throughout the study period. No eDNA for any of the four invasive carp species was detected in water collected from the mesocosms during the three days prior to adding grass carp. Forty-eight hours after grass carp addition to mesocosms, we detected grass carp eDNA in the mesocosm containing 33 grass carp using the LAMP assay. However, we failed to detect any grass carp DNA in the mesocosm containing 3 grass carp with the LAMP assay throughout the study. We analyzed the data using an occupancy model and found that the 500 mL filter samples yielded a higher eDNA capture probability than 50 mL grab samples in the mesocosm containing three grass carp but had similar eDNA capture probability in the mesocosm containing 33 grass carp. Both LAMP and qPCR reliably detected grass carp eDNA 2 days after grass carp addition, but detections were more consistent with qPCR. The LAMP assay may have utility for certain niche uses because it can be used to rapidly analyze eDNA samples and is robust to inhibition, despite having some limitations

Development of a quantitative PCR method for screening ichthyoplankton samples for bigheaded carps

Monitoring ichthyoplankton is useful for identifying reproductive fronts and spawning locations of bigheaded carps (Hypophthalmichthys spp.). Unfortunately, sorting and identifying ichthyoplankton to monitor for bigheaded carp reproduction is time consuming and expensive. Traditional methods require frequent egg-larvae sampling, sorting of all samples to obtain presumptively identified bigheaded carp, and genetic validation of presumptively identified eggs. Quantitative PCR (qPCR) has the potential to streamline this process by identifying samples that likely do or do not contain a target species. Our objective was to develop a genetic screening tool using qPCR with the duplex assays SCTM4/5 and BHTM1/2 to prioritize samples that have a higher likelihood of containing bigheaded carp eggs or larvae. We used tandem ichthyoplankton samples collected for monitoring bigheaded carps in the Upper Mississippi, Illinois, and St. Croix rivers to evaluate the effectiveness of qPCR as a screening tool. Samples with > 10,000 copies of DNA had 100% occurrence of bigheaded carp eggs or larvae in the traditionally sorted samples, whereas samples with This article is publsihed as Fritts, Andrea K., Brent C. Knights, James H. Larson, Jon J. Amberg, Christopher M. Merkes, Tariq Tajjioui, Steven E. Butler et al. "Development of a quantitative PCR method for screening ichthyoplankton samples for bigheaded carps." Biological Invasions (2018). doi: 10.1007/s10530-018-1887-9.</p