P. C. McKinsey
Westinghouse Savannah River Company
Aiken, SC 29808

T. E. Poppy
Oak Ridge Institute for Science and Education (ORISE)
Oak Ridge, TN 37831

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In 1974 the Savannah River Site (SRS) was established as a National Environmental Research Park (NERP) in the United States. NERP provided locations for long-term ecological research investigation. Many of the ecological studies that have been conducted in the past mainly focused on the macroscopic view. The Savannah River Site contains wetlands that are home to many diverse organisms. We conducted a preliminary survey of microbial habitats in order to explore the biodiversity of species-specific symbionts. Bacterial surveys included viable counts, direct counts, isolation, identification, and metabolic profiles.

Recent studies have shown that many invertebrates host species-specific bacteria (Bull 1992). The biodiversity issue will be addressed by asking the following question. Are there bacteriological species that are specific for selected invertebrates? Since species-specific symbionts have been detected with some organisms, it has been assumed that species-specific symbionts exist for most organisms (Bull 1992). This idea has not really been tested. Many arthropods with restricted diets rely on symbiotic associations with microorganisms for full nutrition. The purpose of this survey is to determine whether and how many host-specific bacteria exist in relation to a specific species of insect. This preliminary study of the species-specific bacteria was surveyed in the following species of aquatic insects: Brachycentrus numerosus also known as Caddis fly, Pteronarcys dorsata more commonly known as Stoneflies, and Palaemonetes paludosus also known as a ghost or grass shrimp. These invertebrates were captured from two separate watersheds (Upper Three Runs Creek and Myers Branch) located on the Savannah River Site.

Macro-invertebrate collection. The selected aquatic species were collected from two designated wetland areas located on SRS. The caddis flies were collected during their larva stage of development. There were five individual specimens collected from the Upper Three Runs Creek area and three were collected from the Myers Branch area. They were placed in sterile plastic Nalogene containers with slightly damp paper towels lining the bottom. The shrimp were also collected from both areas and placed in a container with distilled water. There were ten shrimp from Upper Three Runs Creek and nine from Myers Branch. The three stoneflies that were collected were only from Upper Three Runs Creek area. They were placed in a sterile Nalogene container with damp paper towels lining the bottom. The collection of all the specimens was done aseptically using alcohol swabbed forceps. The specimens were brought to the laboratory for species identification (Kondratieff) and weighed and then immediately processed.

Specimen preparation and dilution series. Each set of specimens was placed whole in individual 50 mL sterile tube with 40 mL of FA buffer. Each tube was vortexed for three minutes; and then set for 15 minutes to allow for the insects to soak. The tubes were then vortexed again for three minutes. Next, 30 mL of FA buffer was removed from each tube in order to inoculate duplicate GN2 Biologä plates. Serial ten-fold dilutions were made in phosphate buffered solution to 10^-5. The dilutions were plated onto full strength Peptone, Tryptone, Yeast Extract and Glucose (PTYG) and R2A agar. Then, 10 uL of each solution was placed into the wells of four toxomoplasmosis slides for Acridine Orange Direct Counts (AODCs). The rest of the FA buffer was decanted from the tubes and insects. The insects were mixed with acetone briefly in a glass flask. The acetone was poured off. The insects were rinsed with 50 mL FA buffer three times. The insects were then mixed with sterile 50 mL of sterile FA buffer. Each set was then pureed in a sterile Waring blender container. An additional 50 mL of FA buffer was added to puree and mixed. Next, 30 mL of puree was removed from blender container and used to inoculate duplicate GN2 Biologä plates. Then, 10 mL puree was diluted with 90 mL of sterile FA buffer. This solution was used to inoculate duplicate GN2 Biologä plates. The duplicate BiologÔ plates were incubated at 25^o C for 5 days and then read using the BiologÔ plate reader. Next, 1 mL of puree was removed from blender container in order to form a serial dilution series - 1/10, 1/100, 1/1000, 1/10,000, 1/100,000. The dilutions were then spread onto full strength PTYG and R2A agar plates. The plates were incubated inverted at 25^o C. Then, 10 uL of each pureed solution was placed into the wells of four slides for AODCs. The bacterial counts were counted and then calculated by using the following equations: Total Colony Count/Gram of Wet Weight and Total Colony Count/Insect. The AODC counts were calculated using the same equations. The results of the calculations are listed in tables # 3 and # 4 and compared in # 5.

Isolation, purification and testing of bacteria. From the series of plates that were spread, the bacteria were picked and repeatedly streaked until the cultures were isolated and purified. Each colony of bacteria was measured and a visual description was recorded. The bacteria were then Gram stained in order to determine purity and Gram reaction. Once the bacteria were isolated they were stored on 1% PTYG slants, and also stored in liquid nitrogen (gas-phase) by using the Microbank capsules. The pure Gram-Negative (GN) cultures were then tested for oxidase reaction by using the Oxidase test and Triple Sugar Iron (TSI) slants. The Oxidase test was conducted by aseptically taking a small amount of pure culture and smearing it onto a piece of clean filter paper. A drop of the Oxidase solution was then put on the culture. Within several seconds the results could be observed. The TSI slants were inoculated with pure cultures and allowed several days to grow. The results were observed and recorded to further determine whether the isolate was Gram-Negative Enteric (GN-ENT) or Gram-Negative Non-enteric (GN-NENT) (Biolog).

Classifying isolates using Biologä system. Once the isolates were pure and testing was complete, the isolates were streaked onto Blood Agar Plates. The GN isolates were streaked onto BBLã Prepared Media TSA II with 5% Sheep blood (Difco). The GP isolates were streaked onto BUG (Biolog Universal Growth Media) with 5% Sheep blood. After adequate growth was acquired, the recommended amount of bacteria was then aseptically placed in the BiologÔ inoculating fluid tubes using a sterile swab. The recommended inoculum density for GN-ENT and GN-NENT are 52% and 63% transmittance respectively. The turbidity of the solution was checked using an absorbance/transmittance meter (BiologÔ Inc.). The BiologÔ plates were then inoculated and incubated at 25^o C for several days. The plates were then read in a BiologÔ plate reader using the BiologÔ software. The results were recorded and saved onto a backup disk.

Analysis of Biolog Results and Metabolic Footprints Comparisons. The numerical values of the well optical densities, which signifies any growth of bacteria, for each isolate that was biologed were imported into an Excel worksheet. The values were then replaced by + and – signs in order to be recognized by the JMP statistical software. Any optical density value > 50% greater than the A1 control well was replaced with a + sign. Any optical density value < 50% greater than the control well was replaced with a – sign. This pattern of positive and negative signs was imported into the JMP software (JMP). This software was used to cluster analyze the data. The comparisons were made and put into dendrograms.

During processing the number of specimens and total wet weights were recorded. The solutions of sterile FA buffer used to wash the external bodies of the specimens and the solution of puree in buffer were labeled. The data collected on the day of processing is summarized in Table 1.

Summarized in Table 2 are the BiologÔ results of the duplicate plates using the external washes and internal purees. The positive and negative wells were counted. The duplicates had consistent values. The difference between the caddis fly diluted puree vs. the full strength puree and external from both watersheds would be expected.

The spread plates of the serial dilutions were counted for both bacterial and fungal growth. Table # 3 summarizes the average number of bacterial and fungal colonies for each solution that was plated on both R2A and PTYG media. These averaged numbers were used to calculate the total colony count per total wet weight of all specimens that were processed and then per individual insect.

The acridine orange direct counts were counted and then used to calculate the number of bacterial cells per total wet weight of the specimens collected and per each individual insect. (Table # 4)

Table # 5 summarizes the results of both total colony counts and acridine orange direct bacterial counts per total wet weight of specimens collected and per individual insect. It would be expected to have higher AODC since the acridine orange stains both dead and living cells.

The BiologÔ identification results for bacteria found both internally and externally of each species that are listed in Table # 6. Biolog'sÔ level of identification is listed after each name. The number in parenthesis indicates how many isolates were identified as that species of bacteria.

The data from the metabolic footprint patterns of the BiologÔ plates were compared by imported into the JMP statistical software. A cluster analysis using the Ward method was used to compare how metabolically close these isolates utilize different carbon sources. Dendrogram # 1 compares the internal isolates of all specimens collected at Upper Three Runs Creek. Dendrogram # 2 compares the external isolates of all specimens collected at the UTRC watershed. Dendrogram # 3 compares the internal isolates from the caddis fly and shrimp collected at the Myers Branch watershed. Dendrogram #4 compares the external isolates of the caddis fly and shrimp collected from the Myers Branch watershed. Each dendrogram has the bacteria isolate number and color coded letters to show which type of specimen it was from. The abbreviations are: SF = Stone Fly (orange), CF = Caddis Fly (green) and S = Shrimp (pink).

The important points learned by conducting this preliminary experiment involved the dilution series and the media used to isolate the cultures. Future experiments such as this one should spread plate a 1/10 dilution. This would be 0.1 ml directly from the puree/soak water solution. There was no need for spread plates above the 1/10,000 dilution since no growth occurred at that dilution. The nutrient media that gave better results was the low nutrient R2A. Because of the fungal growth on the spread plates, it would be advised to plate on R2A media with and without cyclohexamide when experiment is repeated. Cyclohexomide would be helpful to deter growth of fungal colonies. The fungal growth on the spread plates made the isolation of the bacterial isolates a little more difficult.

The charts located in the results section are a summary of the preliminary work that was conducted to obtain information on the overall bacterial counts of the internal and external solutions taken from each set of invertebrates. In table # 2 there is a difference in the percentage of positive wells between the caddis fly diluted puree from UTRC and MB. It would be difficult to define a specific purpose to this difference. It maybe contributed to the number of specimens that were processed since number of specimens collected from UTRC and MB were 5 and 3 respectively. The reason that the number of each group of specimens collected from UTRC does not equal the number collected from MB is due to the collectors not being able to find and catch the same number of specimens from second watershed. The UTRC specimens were caught on 2/2/00 and were processed with the understanding that the same number of specimens would be caught from MB on 2/3/00. Being a preliminary study, the experiment was continued even though the numbers of specimens were not equal. The purpose of total bacteria counts from spread plates and acridine orange direct counts would be to see what the recovery rate would be for bacteria isolates for that particular specimen. If the recovery rate were high for a particular specimen, then it would be easier to study any symbiotic bacteria then if the recovery rate were low. In this experiment the recovery rates for each type of specimen were greater than half the direct counts.

During the isolation and purification stage of the experiment there were many bacteria that seemed to live symbiotically. Once they were successfully separated they would no longer grow. Also, there were many bacteria isolates that slowly died out after repeated streaking. These limitations made it quite difficult to isolate each and every bacterial colony found on the spread plates.

Many of the cultures isolated from the internal portions (puree) and external portions of the insects resulted in "No ID." The possible explanation for this could be that the bacteria were not completely isolated or that particular metabolic pattern does not correlate closely enough to the BiologÔ database. The level of identification listed in table # 6 is the percentage of similarity to the bacteria in the BiologÔ database. Identification level > 0.50 would be considered as the ID for that isolate, while a level < 4.9 would result in "no ID" (BiologÔ ).

The useful information that was obtained from the BiologÔ System was its most probable ID and most importantly information about that bacteria’s metabolism. BiologÔ calls this pattern of purple or positive wells the metabolic footprint. The metabolism information was used to construct dendrograms to illustrate how closely related these isolate’s metabolic pathways were to each other. The JMP Statistical software was used to construct these comparisons. The comparisons were made between the specimens that were collected in the same watershed (internal and external). From these dendrograms, it can be seen that some of the isolates have similar metabolic footprints. The dendrograms # 1 – 4 do show that some of the isolates from the individual species have metabolic similarities. The first dendrograms compares all of the specimens internal isolates caught in UTRC. This shows that the stone fly bacteria have a separating group of isolates. The second dendrogram, comparing the internal isolates of the shrimp and caddis fly specimens caught in MB, shows that the majority of the caddis fly isolates and shrimp cluster into their own group. The external comparisons from UTRC and MB would be expected to not have groupings, since all specimens came from the same watershed. The comparisons should have been the different bacteria found between the two watershed for each type of species. Out of the 273 cultures that were isolated only about 70 have been biologed to this point. Therefore, the results that are drawn as this time may not be representative of the actually results when the project is completed and continued in the future.

Dr. Carl Fliermans (SRTC) contributed the project funding and concepts.
Pam McKinsey (SRTC) greatly contributed by creating methods, conducting the bacterial counts and overviewed the projects progress.
Dr. J. Vaun McArthur (SREL) & Dr. Lawrence J. Shimkets contributed the ideas and concepts of the project.
Angela Lindell & Gordie Plague (SREL) conducted the insect collection and identification.
Beth Collup & Jay Wates contributed in streaking plates, assisted in inoculating BiologÔ plates and reading the plates.
Your help was greatly appreciated!

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