2002 Progress Report: A Model for Genetic Diversity Aquatic Insects of the Mobile/Tensaw River Delta

EPA Grant Number: R827072C019
Subproject: this is subproject number 019 , established and managed by the Center Director under grant R827072
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Alabama Center For Estuarine Studies (ACES)
Center Director: Shipp, Robert L.
Title: A Model for Genetic Diversity Aquatic Insects of the Mobile/Tensaw River Delta
Investigators: Brockhouse, Charles L. , McCreadie, John W.
Institution: University of South Alabama
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 2001 through December 31, 2003
Project Period Covered by this Report: October 1, 2001 through December 31, 2002
RFA: Alabama Center For Estuarine Studies (ACES) (1999) RFA Text |  Recipients Lists
Research Category: Targeted Research


The objective of this research project is to identify a limited number of aquatic insects that can serve as model species for genetic diversity studies of the Delta region and similar aquatic habitats. Specific objectives are: (1) collection of samples of four populous, widespread, and highly vagile aquatic insect species; (2) development or adaptation of DNA extraction techniques for each candidate species; (3) identification of 10 or more polymorphic nuclear DNA loci within each candidate species; and (4) preliminary assessment of population structures within the Delta. The results of this genomic inventory, in addition to results generated from companion studies, will be used as a springboard to apply for larger grants (e.g., U.S. Environmental Protection Agency, National Science Foundation) for a complete species inventory and an extensive genomic documentation of Delta insects (companion studies: The Paradox of the Delta, John W. McCreadie and Peter H. Alder, ACES-funded project, May 2000; Total Insect Bio-inventory Project of the Mobile/Tensaw Delta, John W. McCreadie and Peter H. Alder, ACES-funded study, August 2001).

The ideal characteristics for a model species include: high genetic diversity at several loci, good vagility to eliminate the possibility of localized inbreeding through low dispersal levels, significant aquatic stages, significant population sizes to avoid natural/historical causes of inbreeding and to facilitate collecting, and widespread populations throughout the Delta region. Ideally, the species will represent different trophic levels for potential use in biomonitoring studies. The successful identification of model species in this project will lead to subsequent projects, including taxon-specific gene flow studies and biomonitoring of impacted regions.

Progress Summary:

Model Species

We have collected and identified three candidate species for genetic studies: Ranatra nigra (water scorpion), Pelocoris femoratus (nocorid water beetle), and Erythemis simplicicolis (dragon fly). A fourth species, drawn from collections of numerous damselfly species, will be chosen once species identification has been verified through voucher specimens.

DNA Extraction

We have assessed the DNA extraction techniques of Sambrook, Frisch, and Maniatis (1989); Robertson (1993); Brockhouse (unpublished); and Brockhouse, et al. (1993) on R. nigra and P. femoratus. The first three methods are for fresh/frozen specimens. Robertson's method would be particularly valuable because it is extremely rapid. Only the Brockhouse, et al. (1993) methods yielded usable DNA, however. This method involves ethanol fixation of the specimen, followed by prolonged protease/detergent digestion. We have found that alcohol fixation is necessary for both species to prevent contamination of the DNA with large amounts of extraneous co-purifying material, which inhibits subsequent enzymatic steps. Unfortunately, this procedure is time-consuming and laborious. We are continuing to assess alternate, more rapid methods.

Polymorphic DNA Loci

We have cloned four loci from R. nigra: internal transcribed spacers (Brockhouse, et al., 1993), actin intron, elongation factor I intron, and beta-tubulin intron (Palumbi, 1996). To date, we have assessed the internal transcribed spacers for polymorphisms. This locus shows length polymorphisms that are easily scored by agarose gel electrophoresis. Five alleles have been identified to date.

The remaining loci will be assessed for sequence variation by the population heteroduplex assay described in our proposal, augmented by a new technique developed for the identification of human disease-related polymorphisms (Lishanski, et al., 1994). Briefly, polymerase chain reaction (PCR) products from individuals will be mixed, heated, and reannealed to form "heteroduplexes." The MutS protein, purified from Escherichia coli, will be mixed with the PCR products. This protein binds to mismatches and forces the bound DNA molecule to migrate more slowly in polyacrylamide gel electrophoresis. Individuals with differing DNA sequences will be visible by the different migration pattern. The DNA then will be sequenced and the differences identified. We have conducted preliminary experiments with this technique, and it has proven highly satisfactory.


Brockhouse CL, Vajime CG, Marin R, Tanguay RM. Molecular identification of onchocerciasis vector sibling species in black flies (Diptera: Simuliidae). Biochemical Biophysica Research Community 1993;194:628-634.

Lishanski A, Ostrander EA, Rine J. Mutation detection by mismatch binding protein MutS, in amplified DNA: application to the cystic fibrosis gene. In: Proceedings of the National Academy of Sciences, USA 1994;91:2674-2678.

Palumbi S. The polymerase chain reaction. In: Molecular Systematics. 2nd edition. Sunderland, MA: Sinauer Associates, Inc., 1996.

Robertson HM. The mariner transposable element is widespread in insects. Nature 1993;362:241-245.

Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor 1989.

Future Activities:

In the next year, more genes will be cloned from R. nigra, and cloning will soon begin from the other candidate species. Polymorphisms will be discovered using the assay described above, and the alleles will be sequenced at the University of South Alabama DNA sequencing facility. We anticipate that the senior technician soon will be able to join the project.

Supplemental Keywords:

genetic diversity, genomic inventory, water, water use, aquatic ecosystem, coastal ecosystem, coastal environments, environmental chemistry, chemistry, Alabama, AL, aquatic insects, aquatic habitats, DNA, genetic diversity, gene flow studies, biomonitoring, impacted regions, Ranatra nigra, water scorpion, Pelocoris femoratus, nocorid water beetle, Erythemis simplicicolis, dragon fly, polymerase chain reaction, PCR, Escherichia coli, MutS protein, DNA extraction techniques, gel electrophoresis, DNA cloning., RFA, Scientific Discipline, Geographic Area, Water, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, estuarine research, Ecology, Aquatic Ecosystems & Estuarine Research, Chemical Engineering, exploratory research environmental biology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Chemistry, Restoration, State, Aquatic Ecosystem, Monitoring/Modeling, Aquatic Ecosystems, Terrestrial Ecosystems, Aquatic Ecosystem Restoration, Ecological Indicators, plant-animal interactions, coastal ecosystem, water use, aquatic insects, biodiversity, estuaries, watersheds, Alabama (AL), coastal environments, anthropogenic impact, aquatic plants, ecosystem, water quality, estuarine waters, Mobile Delta, adaptation, genetic diversity

Relevant Websites:

http://www.southalabama.edu/aces/ Exit

Progress and Final Reports:

Original Abstract
  • 2003
  • Final

  • Main Center Abstract and Reports:

    R827072    Alabama Center For Estuarine Studies (ACES)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827072C001 Fluorescent Whitening Agents As Facile Pollution Markers In Shellfishing Waters
    R827072C002 Red Snapper Demographics on Artificial Reefs: The Effect of Nearest-Neighbor Dynamics
    R827072C003 Stabilization of Eroding Shorelines in Estuarine Wave Eliminates with Constructed Fringe Wetlands Incorporating Offshore Breakwaters
    R827072C004 Interaction Between Water Column Structure and Reproduction in Jellyfish Populations Of Mobile Bay (SGER)
    R827072C005 Effects of Variation in River Discharge and Wind-Driven Resuspension on Higher Trophic Levels in the Mobile Bay Ecosystem
    R827072C006 Results of Zooplankton Component
    R827072C007 Benthic Study Component
    R827072C008 A Preliminary Survey of Macroalgal and Aquatic Plant Distribution in the Mobile Tensaw Delta
    R827072C009 Fisheries-induced changes in the structure and function of shallow water "nursery habitats": an experimental assessment
    R827072C010 Effects Of Variation in River Discharge and Wind-Driven Resuspension on Lower Trophic Levels of the Mobile Bay Ecosystem
    R827072C011 Evaluation of Alabama Estuaries as Developmental Habitat for Juvenile Sea Turtles
    R827072C012 Effects of Salinity Stress on Natural and Anthropogenically-Derived Bacteria in Estuarine Environments
    R827072C013 The Role of Land-Use/Land-Cover and Sub-estuarine Ecosystem Nitrogen Cycling in the Regulation of Nitrogen Delivery to a River Dominated Estuary; Mobile Bay, Alabama
    R827072C014 Environmental Attitudes of Alabama Coastal Residents: Public Opinion Polls and Environmental Policy
    R827072C015 Synthesis and Characterization of an Electrochemical Peptide Nucleic Acid Probe
    R827072C016 Determinants of Small-Scale Variation in the Abundance of the Blue Crab Callinectes Sapidus
    R827072C017 Effects of Estrogen Pollution on the Reproductive Fitness of the Gulf Pipefish, Syngnathus scovelli
    R827072C019 A Model for Genetic Diversity Aquatic Insects of the Mobile/Tensaw River Delta
    R827072C020 Evaluating Trophic Processes as Indicators of Anthropogenic Eutrophication in Coastal Ecosystems: An Exploratory Analysis
    R827072C021 Effects of Anthropogenic Eutrophication on the Magnitude and Trophic Fate of Microphytobenthic Production in Estuaries
    R827072C022 Characteristics of Ship Waves and Wind Waves in Mobile Bay
    R827072C023 Methods Comparison Between Stripping Voltammetry and Plasma Emission Spectroscopy for Metals in Mobile Bay
    R827072C024 Changes in Water Conditions and Sedimentation Rates Associated With Construction of the Mobile Bay Causeway
    R827072C025 Cold-Induced Hibernation of Marine Vibrios in the Gulf of Mexico: A Study of Cell-Cell Communication and Dormancy in Vibrio vulnificus
    R827072C026 Holocene Sedimentary History of Weeks Bay, AL: Human and Natural Impacts on Deposition in a Gulf Coast Estuary
    R827072C027 Shelter Bottlenecks and Self-Regulation in Blue Crab Populations: Assessing the Roles of Nursery Habitats and Juvenile Interactions for Shelter Dependent Organisms
    R827072C028 Predicting Seagrass Survival in Nutrient Enriched Waters: Toward a New View of an Existing Paradigm
    R827072C029 DMSP and its Role as an Antioxidant in the Salt Marsh Macrophyte Spartina alterniflora
    R827072C030 A Preliminary Survey of Aerial and Ground-Dwelling Insects of the Mobile/Tensaw Delta
    R827072C031 Natural Biogeochemical Tags of Striped Mullet, Mugil cephalus, Estuarine Nursery Areas in the North Central Gulf of Mexico
    R827072C032 Resolution of Sedimentation Rates in Impacted Coastal Environments Using 137Cs and 210Pb Markers: Dog River and Fowl River Embayments
    R827072C033 Investigation of the Use of Pulse Amplitude Modulated (PAM) Fluorometry as an Indicator of Submerged Aquatic Vegetation Health in Mobile Bay
    R827072C034 Influence of Invasive Plant Species in Determining Diversity of Aquatic Vegetation in the Mobile-Tensaw Delta
    R827072C035 The Influence of Shallow Water Hydrodynamics on the Importance of Seagrass Detritus in Estuarine Food Webs
    R827072C036 Food Web Interactions, Spatial Subsidies and the Flow of Energy Between the Mobile Bay Delta and Offshore Waters: A SGER Proposal to the Alabama Center for Estuarine Studies
    R830651C001 Meteorological Modeling of Hurricanes and Coastal Interactions: A Stability Study For Vertical Pressure Levels
    R830651C002 Characterization of Glycoprotein Cues Used by the Parasitic Rhizocephalan Barnacle Loxothylacus texanus To Identify Its Blue Crab Host, Callinectes sapidus
    R830651C003 Survey of Diamondback Terrapin Populations in Alabama Estuaries
    R830651C004 An Assessment of Environmental Contaminant Levels in Water and Dragonfly Larvae Tissues from the Mobile/Tensaw Delta