Bugs in Bugs Analysis of microbiota of Trichnoplusia

Bugs in Bugs: Analysis of microbiota of Trichnoplusia ni Larvae Susan Ha; Co-Authors: Daniela Ochoa and Alberto Hernandez; Advisors: Swapna Bhat, Jeanelle Morgan, and Evan Lampert Department of Biology, University of North Georgia, Gainesville ABSTRACT All multicellular organisms including plants and animals harbor a community of microbes called the microbiota. The gut microbiome plays an important role in digestion, immunity and overall health of an animal. Research has shown that the insect microbiome is associated with resistance to natural invaders, virulence, digestion, and reproductive success. The insect microbiome may also metabolize defensive compounds, and influence the ability of insects to associate with certain host plants. Recent research suggests that hornworm caterpillars (Manduca sexta) have no permanent microbiome; instead acquiring transient microbes from the plants they eat. In this project, students from two courses (BIOL 1108 K and BIOL 2500 L) collaborated to understand the acquisition of gut microbiome of Trichnoplusia ni, a nocturnal cabbage looper or commonly referred to as owlet moths. Biology 1108 K collected fecal samples from T. ni larvae fed on various pre-selected plant diet. The fecal samples were suspended in saline, swabbed on nutrient agar plates, and incubated for 24 h at 37˚C. Bacterial colonies that appeared on these plates were then analyzed and characterized by BIOL 2500 L students. Our group comprising of four students analyzed three bacterial colonies using microbiological techniques learned in BIOL 2500 L. We investigated the colony and cell morphology, Gram reaction, antibiotic resistance and biochemical features of all three bacteria. We identified all three bacterial colonies to be Gram-positive Staphylococcus sp, which is interestingly part of the normal skin microbiota suggesting that humans and insects may share similar microbiota. INTRODUCTION All multicellular organisms including plants and animals harbor a community of microbes called the microbiota. A microbiota comprises of bacteria, archaea, protists, fungi and viruses; and their collective genomes form the microbiome. Specifically, the gut microbiome plays an important role in digestion, immunity and overall health of an animal (Woojun, 2018). The Bugs on Bugs project is a collaborative project involving the students from BIOL 1108 K Principles of Biology II and BIOL 2500 L Microbiology for Allied Health Professions Laboratory. This project was carried out to study the gut microbiome of insects. The gut of insects’ present distinctive environments for microbial colonization. Insects depend on gut bacteria for important functions such protection against natural invaders, digestion, and reproductive success (Engel and Moran 2013, Lewis and Lize 2015). Interestingly, recent research suggests that caterpillars have no permanent microbiome, instead acquiring transient microbes from the plants they eat (Hammer et al. 2017). “Bugs on Bugs” attempted to test a hypothesis that the type of plant a caterpillar gut feeds on does influence gut microorganism species diversity. In this project, the acquisition of gut microbiota by Trichnopulsia ni larvae was studied. The students of BIOL 1108 K collected stool sample from Trichnopulsia ni larvae, suspended the samples in saline, and then transferred onto nutrient agar plates using sterile cotton swabs. As a student of BIOL 2500 L, our goal was to characterize the bacterial colonies obtained by BIOL 1108 K students by using microbiological laboratory techniques. We studied the colony morphologies, determined the Gram reaction, analyzed antibiotic resistance and carried out biochemical testing of the bacterial isolates. MATERIALS AND METHODS 1 RESULTS ISOLATION AND STREAKING: Both of the colonies of BOB_0063 and BOB_0065 are pigmented with a white color, whereas BOB_0064’s colony is pigmented with a yellow color. All three have a circular form with a flat elevation. See Fig. 1 below. Enzyme Amylase Caseinase Gelatinase Substrate Product(s) starch Result Observed BOB # clear zone after adding iodine BOB_0063: BOB_0064: BOB_0065: - sugar units casein clear zone BOB_0063: BOB_0064: + BOB_0065: - liquid after chilling BOB_0063: BOB_0064: BOB_0065: - amino acids gelatin amino acids Figure 1. Pattern of 4 -way or quadrant streak technique and appearance of the three plates forward. GRAM STAIN: BOB_0063 -BOB_0065 have a purple staining, which indicates they have a Gram-positive cell wall. BOB_0063 and BOB_0064 have a coccus (circular or round) shape in a grape-like arrangement, thus being a Staphylococcus organism. The determination of the arrangement of BOB_0065 is unclear due to various distributions of staphlo (cluster), strepto (chain) or diplo (two). See Fig. 2 below. The cell morphology and Gramstain results are summarized in Table 1. Table 2: Summary of biochemical analysis of all three isolates. DISCUSSION In this study, we analyzed gut flora of T. ni fed on the diet of the family Lamiaceae. A summary of the characteristics of all three isolates are presented in Table 3 below. Figure 2. Microscopic view of BOB_0063 (left), BOB_0064 (middle), and BOB_0065 (right). Plate name ALD 4 Plant diet Diet from family Lamiaceae Colony # A, B, and C Date isolated 02/28/2018 BOB # 0063, 0064, and 0065 Colony shape circular Nutrient Agar Plates Color Gram +/- Shape Arrangement Colony edge smooth BOB_0063 Purple Gram-positive Cocci Staphylo- Colony surface smooth BOB_0064 Purple Gram-positive Cocci Staphylo- BOB_0065 Purple Gram-positive Cocci Single Table 1: Summary of Gram stain results and cell morphology. ANTIBIOTIC ASSAY: The isolates were then analyzed for antibiotic resistance. Six commonly used antibiotics such as vancomycin, penicillin, tetracyclin, gentamycin, streptomycin and chloramphenicol were tested. Unfortunately, BOB_0063, BOB_0064, and BOB_0065 did not show any growth on these plates. Hence, our results are inconclusive for the antibiotic assay. See Fig. 3 below. Colony elevation flat Colony color BOB_0063: white BOB_0064: yellow BOB_0065: white Cell shape coccus Cell arrangement Staphlo- Gram Stain Gram-positive Antibiotic resistance Kirby-Bauer test did not work Table 3: Summary of results of BOB_0063, BOB_0064, and BOB_0065. Our results suggest that at least one of the isolates, BOB_0064 may belong to Staphylococcus genus. This is based on Gram staining analysis. Staphylococcus is a Gram positive bacteria which appears as clusters under the microscope. To identify at the species level we will need to conduct several biochemical tests. However, based on visual analysis BOB_0064 could be Staphylococus aureus, which appear as yellow pigmented in nature. Staphylococcus sp are members of the normal microbiota of human skin. The presence of Staphylococcus sp in insect gut is therefore an interesting finding. The other two isolates BOB_0063 and BOB_0065 are Gram positive coccus. It is difficult to speculate their identities due to lack of biochemical testing. Fig. 3. Kirby-Bauer analysis was inconclusive due to no appearance of growth. The results from the antibiotic assay was unsuccessful/ambiguous due to all three plates not having a zone of inhibition as shown in Fig. 3. After redoing the Kirby-Bauer technique for all three plates, the same result appeared – no appearance of zone of inhibition. The same results may indicate the bacteria is not growing properly due to it being old and weak. The antibiotic result was hard to determine due to the no growth. • ISOLATION AND STREAKING • To obtain pure culture and study colony morphology that were streaked onto nutrient agar plates and incubated at 37°C for 24 hours. Bacterial colonies were isolated from the original plate “ALD 4” with a sterile loop and were then observed. LITERATURE CITED 2 • GRAM STAIN • To determine Gram reaction of bacteria and cell morphology (i. e. shape and arrangement). Used three clean slides to prepare the smears and place a loopful of water in the center of the slide. Aseptically touch your loop very lightly to one of the colonies on the streak plate. Let it air dry and heat fix the smear by carefully passing the slide back and forth above the flame. After heat-fixing, place the slides on staining tray. Apply crystal violet to each smear and leave on for one minute. Rinse off with iodine, and then re-apply more iodine and let set for one minute. Rinse off with water. Carefully apply alcohol drop by drop onto the smears, until no fresh stain comes off the smear when a new drop is added (maximum 20 seconds). Rinse immediately with water and blot dry gently with bibulous paper. Apply safranin for 2 minutes, rinse with water, and blot dry. After heat fixing, the slides were Gramstained with the staining reagents: crystal violet, safranin, Gram’s iodine, decolorizing agent such as ethanol, and water and were then observed under a compound microscope. 3 • ANTIBIOTIC RESISTANCE TESTING • To determine/measure antibiotic resistance of each isolated bacteria from three Mueller-Hinton agar plates using Kirby-Bauer disk diffusion with the antibiotics Gentamycin (10 µg), Vancomycin (30 µg), Tetracycline (30 µg), Streptomycin (10 µg), Chloramphenicol (30 µg), and Penicillin (10 µg). Aseptically use a loopful of bacteria from the streak plate and mix into saline tube. Shake until turbidity appears. Aseptically dip one of sterile swabs and completely swab the surfaces of the Mueller-Hinton plate. Due to not having zone of inhibition, the diameter in millimeters was not obtained, thus there was no determination of degree of sensitivity to the antibiotic. 4 • BIOCHEMICAL TESTING • Identification of bacteria species based on enzymatic activities. Aseptically loop the isolated colonies from the streak plates onto a starch agar plate, skim milk agar plate, and nutrient gelatin tubes (3). Inoculate a loopful of bacteria from streak plate in a small, straight line onto the surface of both starch agar and skim milk agar plates. For the nutrient gelatin tubes, push a loopful of the bacteria from streak plate into the medium. After adding iodine to the starch agar plate, the clear zone indicates a presence of amylase in BOB_0064. The lack of clear zone on a skim milk agar plate indicates an absence of caseinase. The nutrient gelatin tubes turning solid after chilling indicates the absence of gelatinase. BIOCHEMICAL TESTING: The isolates were tested for the presence of three extracellular enzymes; amylase, caseinase and gelatinase using starch agar plate (left), skim milk agar plate (middle), and nutrient gelatin tubes (right). The starch agar plate flooded with iodine solution resulted in a dark blue-black color and lack of clear zone, indicating the absence (-) of enzyme amylase in all three BOB. The skim milk agar plate has a clear zone indicating the presence (+) of caseinase in BOB_0064. The solid nutrient gelatin tubes indicated the absence (-) of the enzyme gelatinase in BOB_0063, BOB_0064, and BOB_0065. See Fig. 4 below. Engel, P, and N A Moran. “The Gut Microbiota of Insects - Diversity in Structure and Function. ” FEMS Microbiology Reviews. , U. S. National Library of Medicine, Sept. 2013, www. ncbi. nlm. nih. gov/pubmed/23692388. Engel, et al. “Gut Microbiota of Insects – Diversity in Structure and Function | FEMS Microbiology Reviews | Oxford Academic. ” OUP Academic, Oxford University Press, 1 Sept. 2013, academic. oup. com/femsre/article/37/5/699/542120. Munro, Louise. Concepts in Microbiology Laboratory. Contemporary Publishing Company of Raleigh, Inc. , 2008. Print ACKNOWLEDGEMENTS Fig. 4. Determination of extracellular enzymes of amylase (left), caseinase (middle), and gelatinase (right). I would like to thank the following for their contributions and assistance in the progression of this project: Dr. Bhat, Dr. Morgan, Dr. Lampert, Nou Moua, Daniela Ochoa, Alberto Hernandez, the Department of Biology, and the CURCA Program.