I

Assessment of Microbial Diversity nd Iso].aion oflVlicroor ganisms Assaiatcd wtth Si.1ise-ie

in the American I..ostr iiomaias vneri:mis)

Mary Jo Kirisits, Ph.D.Northwestern University

Joyce M. Sirsoii, Ph.D. Cardatc;Lini .‘ers j Illi,o,.s at L!rbno..-Chainoa :

Microbia1 DiversityJune — July 2000

Abstract

Freshwater and marine decapod crustaceans commonly exhibit exoskeleton degradationwhich is termed shell disease. The disease is an external infection caused by microorganismswhich attack the components of the lobster shell which include chitin, proteins, and lipids.While this disease has been observed for about 170 years in the lobster industry, the most recentand severe occurrences warrant a more extensive investigation. Currently, it is estimated that80% of lobsters in the Buzzard’s Bay, MA have shell disease. From the summer of 1999 to thespring of 2000, every pound system south of Cape Cod has been severely affected by the disease.This infestation has resulted in large-scale loss of primary productivity (up to 35% of the marketvalue) and large culling from the pounds.

Shell-diseased lobsters have been studied for a number of years, but most studies havefocused upon the effects on lobster physiology, rather than focusing on the bacteria speciescausing the infection. The limited literature available concerning isolation and characterizationof bacteria associated with the disease suggest that Vibrio, Aeromonas, and Pseudomonas spp.are suspect organisms. However, detailed analysis of the microbial populations and communitiesassociated with lobster shell disease had not yet been performed.

The current research project focused upon isolation and characterization of bacterialspecies present in wounds of infected lobsters. Enrichment approaches, which focused on themajor components of lobster shell (chitin, protein, and lipid), as well as aerobic and anaerobicisolation techniques were used to culture bacteria which may be participating in the shelldegradation. Pure cultures were isolated from each tested substrate. klentification andcharacterization of the isolates are still under investigation. Additionally, a variety ofmicroscopic examinations were performed on the diseased shells, colonies, and bacterial isolates.Confocal microscopy indicated that the deteriorating shells were covered with biofilms ofvarious types and containing numerous morphologies. Community structure analyses (terminalrestriction fragment length polymorphism and cloning) were attempted to compare the microbialcommunities found in the different appearances of the disease (“cigarette burn” versus “whitespot”) to the microbial communities found in healthy lobster shell and the seawater column.Community analysis has been inconclusive thus far but will be continued.

Using a mixture of the cultures isolated from lobster wounds, it was attempted to infect ahealthy shell. Preliminary evidence suggests that this bacterial mixture caused the appearance ofblack spots on the shell, but the experiment will continue to be monitored. It is undetermined atthis time whether a single isolate can cause shell disease or whether a consortium of organisms isrequired.

Introduction

Freshwater and marine decapod crustaceans commonly exhibit exoskeleton degradationwhich was termed shell disease by Hess in 1937. This is also referred to as rust disease or brownspot. The disease can take three forms: gross anatomy changes due to carapace degradation(lesions, ulcers and erosion), attachment of the outer exoskeleton to the inner soft body (therebypreventing removal of the shell during molting), and small soft patches where chitin and calciumare degraded locally, all of which result in an aesthetically displeasing, often weakened, animal.The blackening of the damaged exoskeleton is due to melanin production, which is an attempt tocontain the damage through clotting (Unestam and Weiss, 1970). Unfortunately, melanizationcan cause problems during ecdysis (process of molting). Aforementioned, the moltshell mayadhere to the new epicuticle, making the lobster unable to completely shed its moltshell.

The disease is an external infection caused by microorganisms which attack thecomponents of the lobster shell which include chitin, proteins, and lipids. However, infectionand degradation are not found equally on different parts of the lobster anatomy. Estrella (1991)noted that shell disease most often occurred on the first pereiopods (claws) followed by theabdomen, carapace, and legs. Estrella (1991) suggested that the high incidence of shell diseaseon the claws could be due to its propensity for abrasion and its frequent contact with sediments.

While this disease has been observed for about 170 years in the lobster industry, the mostrecent and severe occurrences warrant a more extensive investigation. In 1991, 51% of BuzzardsBay lobsters had shell disease (Estrella, 1991). Currently, it is estimated that 80% of lobster inthe Bay have shell disease (Syslo, 2000). From the summer of 1999 to the spring of 2000, everypound system south of Cape Cod has been severely affected by the disease, particularly thedifficult molting variety (Enos, 2000). This infestation has resulted in large-scale loss of primaryproductivity (up to 35% of the market value) and large culling from the pounds. Shell-diseasedlobsters can be sold commercially, but they command a reduced price, and are usually used forcanned meat. However, Floreto et at. (2000) determined that the nutritional value of meat from adiseased lobster is similar to that from a healthy lobster, in terms of essential fatty acids.

The crustacean cuticle has been described as consisting of four layers (Dennel, 1960).The outer layer, the epicuticle, is a thin layer consisting of proteins and lipids. Beneath theepicuticle are three chitinous layers which are called the exocuticle, calcified endocuticle, andthe non-calcified endocuticle. The epicuticle can be damaged through mechanical abrasion orenzymatic attack (proteases or lipases). If the epicuticle is repaired in a timely fashion, thechitinous layers are not subject to attack by chitinoclastic bacteria. Stewart (1980) noted severalfactors that could prevent or reduce the efficiency of epicuticle repair such as poor diet, repeatedabrasion (dredging, fighting, crowded impoundments), and enzymatic attack. It has beensuggested that lipolytic bacteria may degrade the epicuticle, thereby providing an entry for thechitinoclastic bacteria to reach the chitinous layers (Baross and Tester, 1975).

There are a number of factors that affect molting frequency and therefore the incidence ofshell disease: size, sex, and ovigerous condition of the lobster. Larger lobsters molt lessfrequently. Non-ovigerous females (those not carrying eggs) molt less frequently than males.Ovigerous females molt less frequently than non-ovigerous females because molting is delayed

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until after the eggs are hatched. As the time between molts increases, the lobster shells may havelonger contact times with the causative agent(s) of shell disease. Thus, ovigerous female lobstersshow the greatest incidence of shell disease.

Shell disease is contagious when lobsters share a seawater habitat and have repeatedphysical contact (Fisher et al., 1978). Shell disease may be associated with municipal andindustrial pollution which cause high organic loading. For instance, Estrella (1991) notes thatpolychiorinated biphenyls, heavy metals, and hydrocarbons are prevalent in Buzzards Bay wherethe incidence of shell disease is quite high. Thus, pollutant minimization may help to decreasedisease occurrence in the wild. However, more specific measures may be taken to reduce theincidence of shell disease in lobster impoundments. For example, to treat affected lobster larvae,Fisher et al. (1976) suggest that the larvae be dipped in 20 mg/L malachite green dye solution foreight minutes every other day during the larval period. Getchell (1989) notes that impoundmenthygiene may be improved by removing wastes and disinfecting influent water through ultravioletirradiation. Since chitinoclastic bacteria may reach the chitinous shell layers through a wound, itis also important to reduce wounding. Reducing the number of lobsters per volume, decreasingthe impoundment time, and proper diet can decrease lobster wounding (Getchell, 1989).

Previous research on shell disease has been mainly limited to observations focused on thelobster physiology (Prince, 1995; Floreto et al., 2000). Fisher et al. (1978) reviewed the sixknown microbial diseases of cultured lobsters, which include: shell disease, Gaffkemia,microbial epibiont, Lagenidium, Haliphthoros and Fusariu,n diseases. The chitinoclasticmicroorganisms of shell disease do not penetrate into soft tissues but may provide a portal forsecondary invaders. While most investigations have used adult lobsters, death is more frequentin larval stages, presumably due to thinner exoskeletons (Fisher et al., 1976). Microbiologicalobservations have been limited to mainly microscopic examinations using light microscopes, xray analysis, and scanning electron microscopy (Bayer, 1989). Literature concerning isolationand characterization of chitinoclastic bacteria have suggested that Vibrio, Aeromonas, andPseudomonas spp. are suspect organisms (Getchell, 1989; Malloy, 1978; Prince, 1997).However, detailed analysis of the microbial populations and communities associated with lobstershell disease has not yet been performed. With the advent of molecular techniques enablingmore detailed examinations, it is paramount to the lobster industry to investigate not only thecausative agents, but also the potential opportunistic colonizers which may exacerbate thedisease. Additionally, a more detailed identification of the pathogenic organisms involved maylead to new treatments or direct future research in abatement and prevention techniques.

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Objectives

Since interest in the causative agent(s) of shell disease is growing, the main goal of thisproject was to examine the organisms present in the wounds of lobsters with shell disease.Specific goals were as follows:

• Culture organisms from lobster wounds based on their ability to degrade chitin, proteins, orlipids.

• Isolate and characterize pure cultures from mixed community obtained from wounds.

• Determine if pure cultures of organisms isolated from lobster wounds can be used to infect ahealthy shell.

• Compare microbial communities present in the seawater column and on a healthy lobstershell to the microbial communities present in the “cigarette burn” and “white spot”appearances of shell disease.

• Isolate DNA and use clone libraries to assess the identities of organisms associated with shelldisease wounds.

• Visualize the bacterial biofllms that grow on lobster shells.

Materials and Methods

LOBSTERS

Procurement

Four female lobsters (I healthy and 3 diseased) were obtained from Mike Syslo of theLobster Hatchery on Martha’s Vineyard. The healthy lobster was obtained from VineyardSound, and the three diseased lobsters were obtained from about eight miles south of No Man’sIsland. The diseased lobsters had shell disease that affected the pereiopods, carapace, andabdomen; this disease manifested itself in the appearance of cigarette burns. Another diseasedfemale lobster was obtained from Dr. Rainer Voigt of the Marine Biological Laboratory. Thislobster had many white patches, especially around the first pereiopods (claws). A summary oflobster information is shown in Table 1.

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Table 1. Summary of Lobster Information

Lobster ID Source Disease Appearance Approx. age (yrs)A 8 mi. south of No Man’s Island Cigarette burns 7B 8 mi. south of No Man’s Island Cigarette burns 7C 8 mi. south of No Man’s Island Cigarette burns 7D New Bedford, MA pound White patches 8E Vineyard Sound Healthy - control 7

Housing and Maintenance

The lobsters were housed in 54 x 35 x 35 cm tanks. Each lobster was placed in a separatetank, except Lobsters B and C shared a tank. Fresh seawater (22 °C) was continuously pumpedthrough the tanks at a rate 5.3 L/min. The lobsters were fed once or twice a week with fresh orfrozen squid tentacles from the Marine Resource Center.

SAMPLING FROM THE LOBSTERS

Lobsters were removed from the tanks, rinsed thoroughly with sterile seawater, and driedwith Kimwipes® prior to sampling. Samples were collected from the lobsters by swabbing andscraping the shell with either a sterile swab or scalpel.

LIQUID ENRICHMENT MEDIA

Synthetic Seawater Media

Four synthetic seawater media were designed for aerobic liquid enrichments: ball-milledchitin (Sigma Chemical Co., St. Louis, MO) as the sole carbon and nitrogen source, Bacto®

Peptone (Difco Laboratories, Detroit, MI) as the sole carbon and nitrogen source, Tween® 80 (J.T. Baker Chemical Co., Phillipsburg, NJ) as the sole carbon source with ammonium as thenitrogen source, and a medium containing a mixture of chitin, peptone, and Tween® 80. Themedium contained (per liter of medium): 20 g NaCI, 3 g MgCl2•6H20,0.15 g CaC122H2O, 0.2g KH2PO4,0.5 g KC1, 1 mM Na2SO4,4 g of ball-milled chitin OR 4 mL Tween® 80 OR 4 g ofpeptone OR all three, 1 mL l000x trace elements solution, 1 mL 1000x 12-vitamins solution, 1mL vitamin B12 solution, 5 mM Hepes buffer, and 2 mM NaHCO3. The Tween® 80 mediumalso contained 0.5 mM N1-I The pH of all media was adjusted to 8 using 10 N NaOH. Thetrace elements solution consisted of (per liter): 5200 mg EDTA adjusted to pH 6.0 with NaOH,2100 mg FeSO4’7H20, 30 mg H3BO3, 100 mg MnCI24H2O, 190 mg CoC12’6H2O, 24 mgNiC12•6H2O, 2 mg CuCl2•2H20, 144 mg ZnSO47H2O, 36 mgNa2MoO4•2H2O,25 mg sodiumvanadate, 6 mgNa2SeO35H2O,8 mgNa2WO42H2O. The l000x 12-vitamins solution consistedof (per 100 mL): 100 mL of 10 mM phosphate buffer (pH 7.2), 10 mg riboflavin, 100 mgthiamine•HC1, 100 ing L-ascorbic acid, 100 mg D-Ca-pantothenate, 100 mg folic acid, 100 mgniacinamide, 100 mg nicotinic acid, 100 mg 4-aminobenzoic acid, 100 mg pyridoxineHCl, 100

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rng lipoic acid, 100 mg NAD, and 100 mg thiamine pyrophosphate. The l000x vitamin B12solution consisted of 100 mL DDW and 100 mg of cyanocobalamin.

The carapace of Lobster B was scraped and a portion of the scrapings was placed into 1.0mL of each of the four media. Medium (4.5 mLs) was added to sterile 18x150-mm tubes whichwere capped with sterile Morton closures. Twenty tubes were prepared so that a ten-dilutiontube experiment could be run in duplicate. 0.5 mL of the stock inoculum was transferred into thefirst tube, and 1:10 dilutions were made with the remaining nine tubes. The liquid enrichmentswere incubated aerobically at room temperature on a shaker table. Terminal dilutions stillshowing activity were used for DNA extractions (Table 2, samples 14-17).

SOLID MEDIA

Solidified Synthetic Seawater Media for Enrichment Isolations

The synthetic seawater media used for the liquid enrichments (chitin, peptone, Tween®80, and a mixture of chitin, peptone, and Tween® 80) were also used to prepare agar media.Twenty g of washed agar was added per liter of medium. These plates were inoculated from theliquid enrichments, using the highest dilution that still showed turbidity. 0.1 rnL was taken fromthe terminal dilution, and 1:10 dilutions were performed into sterile 2 mL eppendorf tubescontaining the appropriate liquid medium. 0.1 mLs from the 1:100,000 and 1:1,000,000 tubeswere spread onto the agar plates and incubated aerobically at room temperature. Isolatedcolonies were streaked twice for purity.

Aerobic Seawater Media to Isolate Chitin-Degraders

Basal seawater medium contained I L freshly collected seawater amended with 15 gwashed agar and was autoclaved. Plates were poured and allowed to harden before the chitinoverlay was added. The chitin layer contained 4.0 g chitin (either ball-milled or unbleachedflaked), 20 g washed agar, 0.70 g K2HPO4,0.50 g MgSO4•7H20,0.30 g KH2PO4,and 1.0 mLtrace mineral solution per 500 mL distilled deionized water. This was adjusted to pH 8.0 ± 0.2 at25 °C. After autoclaving, the medium was cooled to 55°C and poured in a thin layer over thehardened basal media. Plates were allowed to harden and then stored at 5°C until use. Platesaged for at least 24 hours prior to use to allow a gradient to form between the seawater layer anddistilled deionized water layer.

Fresh swabs from the lobsters were spread in duplicate over the aerobic chitin medium toform a lawn and incubated aerobically at either ambient room temperature or chilled to 9°C.

Anaerobic Seawater Media to Isolate Chitin-Degraders

Anaerobic media was prepared as described above for the chitin layer of the aerobicmedia with the following exceptions: 500 mL fresh seawater was substituted for distilled water,250 jiL resazurin (1 % stock) and 0.25 g cysteine HCI was added to media. The medium wasboiled to dissolve the agar and then gassed with a mixture ofN2/C02 to reduce the dissolved

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oxygen concentration. The medium was dispensed into baich tubes (10 mL) and sealed prior toautoclaving. After cooling to 55°C, the tubes were inoculated under gas with samples fromlobsters described below, resealed and gently mixed without introducing bubbles within themedium. The roll tube method was applied to the sealed tubes to produce a thin solid layer ofchitin medium to the inner surface of the balch tubes. Chitin degradation should be easilyobservable via clearing zones within the thin layer formed.

The remaining anaerobic media was used in a series of pour plates which were inoculatedwith material scraped from the diseased lobsters. The pour plates were permitted to go aerobicon the surface to form an oxygen gradient. The plates were then sealed with parafilm andincubated at room temperature.

Characterization of Chitin-Degrading Isolates Media

Bacterial isolates obtained from solid media were streaked for purity and transferredtwice. After purification, isolates were inoculated into a variety of motility agar stabs. Basalstab media contained 0.35 g K2HPO4,0.25 g MgSO4•7H20,0.15 g KH2PO4,and 0.5 mL tracemineral solution added to 500 mL of either fresh seawater or distilled deionized water. Themedium was split into 250-mL portions, and each portion received 1 g ball-milled chitin. Each250-mL portion was mixed and split into 125-mL portions and 1.25 mL 1M MOPS buffer (pH7.0) was added to one each of the final flasks. Total media was (8) 125-mL aliquots of twowater types, half containing chitin additions and half containing buffer additions. All wereadjusted to pH 8.0 ± 0.2 at 25 °C with 1 M NaOH. Solidifying agent was 0.2 g agarose per flaskwhich was added prior to autoclaving. After the media cooled to 60°C, 10 mL aliquots weredispensed aseptically into test tubes which were then vortexed and placed in an ice bath toharden immediately.

DNA EXTRACTION

Samples 1-8 (Table 2) were collected from each lobster by scraping the outer shell with asterile scalpel. Lobsters were removed from each tank, rinsed thoroughly with sterile seawaterand dried with Kimwipes® prior to scraping. The scraped material was collected and added to aDNA extraction tube (MoBio Inc., Solana Beach, CA) until visually turbid. The accumulatedscum layer from the tank housing lobsters B and C (sample 9) was also used for DNA extraction.A sterile pipet was used to harvest 250 mL of the scum layer directly into a vacuum-filterapparatus containing a 0.22-pm filter. The filter was cut into squares with a sterile razor blade ina sterile petri dish and then placed in a DNA extraction tube. For DNA from seawater (samples10-13, Table 2), fresh seawater was filtered through a 125-mm Whatman #1 filter in a Buchnerfunnel to remove gross particulates. Then one liter of the filtrate was vacuum-filtered through a47-mm nylon 0.22-tm filter (MSI, Inc., Westboro, MA). As described previously, the filter wascut into pieces and placed into a DNA extraction tube. This procedure was performed for fourfilters, and each filter was placed into a separate DNA extraction vial. DNA was also extractedfrom the terminal dilution tubes for the liquid enrichments containing chitin, peptone, Tween®80, and a mixture of the three (samples 14-17, Table 2). One mL from each terminal dilutionwas pipetted into the DNA extraction vial. DNA was extracted according to manufacture’s

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directions with the following exception: tubes were placed in the bead beater (BioSpec Products,Akron, OH) for 45 seconds, rather than shaken for 10 minutes on a tabletop vortex. DNA qualitywas assessed using a 1 % LE agarose gel (lx TBE buffer), run for 35 mm at 150 V, and stainedwith GeiStar® (BMA, Rockland, ME).

Table 2. Sample Identification for Isolations and DNA Extractions

Sample Lobste Description DNA PCRID r ID Extracted Amplification*

1 B Carapace No -

2 A Carapace Yes A,B,T3 B Carapace Yes A, B, T4 C Carapace Yes A,B,T5 C 1st pereiopod Yes A, B, T6 C Abdomen Yes B7 D White spot, under lstpereiopod Yes B, T8 D Tan spot on right 1St pereiopod Yes B9 Tank Filter with 250 mL scum layer Yes B, T

B/C10 Filter with 1 L seawater Yes T11 Filter with 1 L seawater Yes T12 Filter with 1 L seawater Yes T13 Filter with 1 L seawater Yes14 B 1:100,000 dilution Yes T

15 Yes T

16 Yes T

(chitin enrichment)B 1:1,000,000 dilution

(peptone enrichment)B 1:1,000,000 dilution

(Tween® 80 enrichment)1:100,000 dilution(chitin, peptone,enrichment)

17 B Yes TTween® 80

* A = Archaeal primers; B = Bacterial primers; T Bacterial primers for T-RFLP

PCR AMPLIFICATION

PCR of 16S rDNA Gene

PCR was performed on extracted DNA samples using both bacterial and archaeal specificprimers. Bacterial 16S amplification used primers S-D-Bact-0008-F-20 and19 and archaeal 16S amplification used primers S-D-Arch-2l-F and S-D-Arch-958-R. PCRreactions contained: 5 jiL lOx reaction buffer; 4 iL 25 mM MgCl2; 2 iL 2.5 mM dNTP mix; 2iL each forward and reverse primer (30 pMol); 2 jiL sample DNA (‘.- 125 ng); 0.5 jiLArnpliTaq DNA polymerase (5U/jiL) (Applied Biosystems, Foster City, CA); and sterile distilled

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deionized water to a total volume of 50 jiL. A 25-cycle PCR program was used: 95°C initialdenaturation (5 mm), 95°C denaturation template (30 sec), 55°C annealing temperature (30 sec),72°C extension (1 mm), and 72°C final extension (7 mm) for both reaction sets. PCR productwas assessed on 1% LE agarose gel (lx TBE buffer) for 35 mm at 150 V and stained withGelS tar®.

Cloning

PCR products ( 1400 Kb) that were obtained from DNA extracts of lobster shells werecloned using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) following manufacture’sprotocols. Samples which had sufficient PCR product were cloned. Clones were plated on LBagar (1 % tryptone, 0.5 % yeast extract, 1 % NaCI, pH 7.0) with 50 jig /mL kanamycin addedafter autoclaving. X—gal (40 mg /mL) was added (40 jiL) and spread upon surface after plateshardened. Both 50 and 80 jiL aliquots of each cloning reaction were plated to ensure adequatecoverage. Clones indicating positive for DNA inserts were selected and tested with PCRamplification using M13 primers specific for the vector insert region. Products were analyzed ona 2% agarose gel in 1X TBE buffer at 130V for 2 h and stained with GeiStar®.

Terminal Restriction Fragment Length Polymorphism (T-RFLP)

PCR was performed on extracted DNA samples with bacterial primers - S-D-Bact-0008-F-20 and The forward primer was labeled with a fluorochrome. PCRreactions contained: 5 jiL lOx reaction buffer, 4 jiL 25 mM MgCI2,2 jiL 2.5 mM dNTP mix, 21iL each forward and reverse primer (-30 pMol), 1 jiL sample DNA, 0.5 jiL AmpliTaq DNApolymerase (5U/jiL), and 33.5 jiL sterile distilled deionized water for a total volume of 50 jiL. A25-cycle PCR program was run which consisted of: 95°C initial denaturation (5 mm), 95°Cdenaturation template (30 sec), 55°C annealing temperature (30 sec), 72°C extension (1 mm),and 72°C final extension (5 mi. PCR product was assessed on a 1% LE agarose gel (lx TBEbuffer) for 35 mm at 150 V and stained with GeiStar®.

Only samples that showed PCR product were digested. The digestion reaction consistedof the following: 10 jiL PCR product, 2 jiL lOx Buffer A, 2 jiL BSA, 1 4L of RSA-I, and 5 jiLof sterile distilled deionized water. The samples were incubated in a water bath at 37 °C for twohours. 10 jiL of the digestion was sent for T-RFLP analysis (Accugenix, Newark, DE).

INFECTING A HEALTHY SHELL

Fresh rnoltshell was obtained and cut into small pieces with a sterile blade. Each piecewas sterilized with ethanol and placed into a sterile petri dish. One dish was filled with themedium containing the mixture of chitin, peptone, and Tween® 80, and an X was scratched onthe shell with a sterile blade. The four pure cultures were swabbed into the X. The last dish wasfilled with the medium containing the mixture of chitin, peptone, and Tween® 80, an X wasscratched on the shell with a sterile blade, but no microorganisms were inoculated to this dish(control). The shells were monitored for visual appearance of shell disease under the dissectingmicroscope.

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BIOFILM DEVELOPMENT

Using fresh, non-diseased lobster moltshell, lxi cm pieces were cut with a sterile razorblade. A small hole was punched in the corner of each piece, and the pieces were threaded withfishing line. The line was suspended in the scum layer at the top of the tank housing Lobster A.Three of the pieces were roughened with a sterile needle, and three pieces were left smooth.

During the duration of the experimental period, lobster C went through the molt process.Upon successful ecdysis, the shell remains were removed from the tank and frozen for futureanalysis of bioflims associated with the advanced stages of disease. Biofilms were visualized asdescribed below.

MICROSCOPY

Healthy and diseased lobsters were macroscopically photographed using a dissectingscope (Zeiss, Stemi 2000-C) and MetaMorph software (Version 4.1.4)

Wet mounts of microorganisms swabbed from healthy and diseased lobsters wereexamined using a Zeiss epifluorescence microscope (MC 80 DX). Liquid enrichments and purecultures were examined with phase contrast microscopy. Some isolates were also stained with0.01 % acridine orange for fluorescence microscopy or with methylene blue for lightmicroscopy. Fluorescence images were captured using I OOx lens and DAPI-5 filter set.

Lobster moltshell suspended in the scum layer of Tank A were examined for biofilmdevelopment using a Zeiss laser scanning confocal microscope (LSM 510). Moltshell wasstained in 0.01 percent acridine orange for 1 minute and then rinsed with sterile seawater. Thetop of the moltshell was placed on a coverslip slide. Two-inch lengths of transfer pipet tips wereadhered to the length of the slide with vacuum grease in order to reinforce the slide. Vacuumgrease was also used around the edges of the moltshell to adhere it to the slide.

The diseased carapace of a lobster moltshell (lobster C) was cut into 2 cm x 2 cm piecesand immobilized inside a disposable petri dish using melted paraffin. Pieces were stained forepifluorescence viewing by submerging pieces in 9 ml of stain. Each piece was staineddifferently: 1) non-stained control (flooded with sterile seawater); 2)1/50 dilution of DAPI (40

mg/ml stock); 3)1/10 dilution acridine orange (0.01% stock); 4)1/100 dilution SYBR Green®(I000X stock) mixed 1:1 with distilled deionized water. Pieces were stained for 10 minutes inthe dark, rinsed with distilled deionized water briefly and flooded with anti-fade solution andkept in the dark until viewed. Images were collected using a Zeiss LSM-510-NLO 2-photonconfocal laser scanning microscope using Axioplan 2 imaging software. Using a 40X

water/glycerol wet immersion lens and an 800 or 488 nm 2 scan, Z stacks of 70 1iM werecollected with either 60 to 266 stack passes.

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Results and Discussion

GROSS ANATOMY AND VISUAL ASSESSMENT OF SHELL DISEASE

The gross anatomy of each lobster was documented upon attainment (Figure 1). Areas ofthe shell typically affected by shell disease were examined, noted, and photographed when shelldisease was found (Figures 2 and 3). Individual wounds were examined and typical healthymorphology (Figure 4), as well as lesions (Figures 5 and 6) were photographed using a dissectionscope. Lobsters A, 13, and C, had relatively similar disease etiology in that both the 1st pereiopodswere affected as well as the carapaces and abdomens. Additionally, disease was not noted uponthe remaining pereiopods, with the exception of lobster C, who was showing initial signs of thedisease. Lobsters were separated into tanks based upon the severity of the disease or by diseasemorphological differences. During the experimental period, lobster C went through the moltcycle as described previously. Due to the severity of disease exhibited by this specimen, themolt was incomplete and the right 1st pereiopod was lost at the axial point of connection to thecarapace. Additionally the minor clasper of the left 1st pereiopod was also lost at the midpoint.‘While the molt resulted in a lobster free of disease, there is a distinct disadvantage for this animalto survive unaided until the lost appendages can be re-grown.

LIQUID ENRICHMENTS

There are three main components to the lobster shell: chitin, proteins, and lipids. Theouter layer, called the epicuticle, is a non-chitinous layer composed of proteins and lipids. Thenext three layers, which are the exocuticle, calcified endocuticle, and non-calcified endocuticle,contain chitin. Since lobster shell disease usually penetrates to the chitinous layers, theenrichments included chitin, proteins, and lipids. Ball-milled chitin was used in the enrichmentsso that the surface area available to the microorganisms would be high. Bacto® Peptone, whichconsists of partially digested proteins, was chosen as the source of peptides. To assess lipid use,Tween® 80 was chosen because it contains a fatty acid side group (oleic acid).

The dilution-to-extinction technique was employed to enrich for microorganisms presentin the lobster shell wounds. This technique, consisting of progressive dilutions, was used byJackson et al. (1998) who was able to culture different microorganisms at different dilutions.For example, microorganisms that grow very well in the culture medium will be selected for atlower dilutions; they will be able to outcompete organisms that are originally present in highernumbers but are not well-suited to the chosen culture medium. On the other hand,microorganisms that were abundant in number in the original culture will be selected for athigher dilutions since the less abundant organisms will be diluted out of the culture. Thus, theuse of the dilution-to-extinction technique is essential for enriching the numerically dominantmicroorganisms present in the lobster wounds.

After three days, the liquid enrichment tubes were observed for turbidity. The highestdilution still showing turbidity was used for microscopy. Table 3 lists the terminal dilutionshowing turbidity for each liquid enrichment and the morphologies present in each enrichment.

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Figures 7-10 are phase contrast micrographs showing dominant morphologies in the liquidenrichments. Figure 7 shows the fat curved rods and short thin rods that were dominant in thechitin liquid enrichment. Figure 8 shows the rods that were dominant in the peptone liquidenrichment; some of the rods occurred as single rods and others were clustered. Short fat rodswere the dominant morphotype in the Tween® 80 liquid enrichment (Figure 9). Rods were alsothe dominant morphotype in the liquid enrichment consisting of a mixture of chitin, peptone, andTween® 80; the single rods and clusters of rods look very similar to those seen in the peptoneenrichment (Figure 10). Additionally, Figure 10 shows a long spirillum that was seen in themixture liquid enrichment. Although the spirilla were not the dominant morphology, quite a fewwere present in this enrichment.

Table 3. Results of Liquid Enrichments

Enrichment Terminal Dilution MorphologiesWith Turbidity (* dominant)

Chitin 1:100,000 Rods (motile and *ic motile)Fat curved rods (*nonmotile)

Long thin rods (motile)Peptone 1:1,000,000 Single rods (motile and *nonmotile)

*Clustered rodsLong thin rods (non-motile)

Tween® 80 1:1,000,000 Rods (*motile and non-motile)Rods (*motile and *non motile)

Mixture 1:100,000 *Clusters of rods(chitin, peptone, Tween® 80) Long thin rods (motile)

Long spirilla (very motile)

ISOLATIONS FROM LIQUID ENRICHMENTS

Material from the terminal dilutions of the liquid enrichments still showing activity wasused in plating experiments. The plates consisted of liquid media that had been solidified withagar. In this manner, one pure culture was obtained from each of the liquid enrichments. Table4 summarizes the morphology and motility of the pure cultures. Figures 11-14 show phasecontrast micrographs of the pure cultures.

Table 4. Pure Cultures Isolated from Liquid Enrichments

Enrichment Colony Morphology Organism Morphology Motility

Chitin Clear Curved rod Very motilePeptone White Rod Motile (slow)

(single and clustered)Tween® 80 Clear surrounded by Rod Motile

halo of whiteprecipitate

Mixture White Rod Non-motile(chitin, peptone, Tween® 80)

12

The isolates that were cultured on the chitin plates grew quite slowly. After one week ofgrowth, faint clearing zones in the chitin were able to be seen. By contrast, the isolates culturedon peptone and the mixture of chitin, peptone, and Tween® 80 grew much more quickly;individual colonies were observed after one or two days of incubation. The colonies on the agarwith Tween® 80 as the sole carbon source grew more slowly. Colonies grew after four days, andthe plates had an interesting appearance. Clear colonies were surrounded by a halo of whiteprecipitate which is shown in Figure 15. If the oleic acid side group present in Tween® 80 iscleaved from the molecule, due to the action of a class of enzymes called lipases, it can reactwith calcium present in the medium to form calcium soap precipitate. Agar plates supplementedwith Tween® 80 and calcium are often used to assay for liapse activity, and the presence of thewhite precipitate halo surrounding the colony indicates that the organisms are secreting lipase(Gerhardt et al., 1994).

INFECTING A HEALTHY SHELL

The four pure cultures shown in Figures 11-14 were used in an attempt to infect a healthymoltshell with shell disease. After four days of incubation, the control shell (not inoculated) andthe test shell (inoculated with the four pure cultures) were examined under the dissectingmicroscope. As expected, no colony development or degradation was observed on the sterilecontrol shell (Figure 16). The black spots observed on the control shell were part of the normalcoloration of the shell. On the test shell (Figure 17), no colony formation or degradation wasobserved on the X scratched into the shell which exposed the chitinous layers. This is likely dueto the slow growth of chitin-degraders, which probably had not grown much during the four-dayincubation period. On the other hand, large black spots can be seen on the test shell, and it ispossible that these are the beginning of shell disease. Note that these larger black spots are closeto the X where the pure cultures were swabbed. The black spots may be due to the lipasesecreting organisms cultured on the Tween® 80 medium, since this organism could break downthe lipids on the epicuticle of the lobster shell. This shell will continue to be monitored todetermine if the black spots continue to grow and cause pitting of the shell.

T-RFLP

Digested PCR amplicon was sent for T-RFLP analysis for samples 2, 3, 4, 5, 7, 9, 11, 12,14, 15, 16, and 17. A description of each of these samples is in Table 2. Sample 17 was sent toAccugenix but was not run in the T-RFLP analysis for an unknown reason. Figure 18 shows thebanding pattern for the T-RFLP samples. The red bands are from standards, and the blue bandsare from the sample PCR amplicon. Only sample 16, from the Tween® 80 enrichment, showed adistinct banding pattern along the length of the gel. All of the other samples only showed abright band at the bottom of the gel and no bands along the length of the gel. PCR amplicon wasobserved for all of these samples, so lack of PCR product is not the cause of this lack of banding.Rather, it is likely that the length of the terminal restriction fragments were short causing them totravel rapidly through the gel; thus, this led to bright bands at the bottom of the gel.

13

Based on this T-RFLP analysis, nothing can be said about differences in the microbialcommunities of seawater, “cigarette bum” shell disease, and “white spot” shell disease.However, the PCR amplicon for these samples will be digested with a different restrictionenzyme, resulting in different terminal fragment lengths, and again be analyzed on a gel.

BIOFILM DEVELOPMENT

One of the moltshell squares that was suspended in the scum layer of the tank housingLobsters B and C was removed and stained with acridine orange for confocal microscopy. Themoltshell was soft, indicating degradation of shell components. Figure 19 shows a picture of thebiofilm that had developed on the shell. Note the large number of rod-shaped organisms that arepresent in the biofilm. Figure 20 shows spirilla that were also present in the bioflim; theyseemed to be quite a bit shorter than the spirilla that were observed in the enrichment containinga mixture of chitin, peptone, and Tween® 80.

ISOLATION OF CIIITINOCLASTIC BACTERIA USING SOLID MEDIA

Swabbed Plates

Swabbed chitin plates developed confluent growth of several colony morphologies after aweek. Ten different morphologies were selected from various lobster samples and transferred tofresh plates (Table 5). The transferred isolates again only showed growth after a week and didnot show any chitin degradation until the 101h day of incubation (Figure 21). Half of thetransferred isolates did not show any sign of growth even after 14 days incubation. Of those thatdid grow, the ones from the control lobster and lobster B had short thick non-motile rods.Isolates from lobster C-abdomen were separated to the point of a co-culture containing very shortthick ovoid rods demonstrating gliding motility and spirillum what is suspected to be a memberof the genus Saprospira. The suspect Saprospira is a long helical spiral which contains darkinclusion bodies and forms spheroplasts upon aging of cells (Figure 22). Careful examination ofgrowth conditions and cell descriptions listed in Bergey’s Manual suggest this may be a newmember of the genus based upon its chitin degradation capabilities (Figure 21). Insufficient timeremains in course to attempt further classificationlidentification.

14

Table 5. Characteristics of Colonies from Solid Media

Sample I.D. Colony Morphology Cell MorphologyA-i White, convex, latticed No growthA-2 Pink, umboed, convex No growthB-i Beige, wrinkled, irregular Short, fat rods, non-motileB-2 White, smooth, entire, convex Short rods, non-motileC-i White, mucoid, spreader Long thin rods, motileC-2 Pink, entire, concave, hardened No growthC-3 White, umboed, convex, spreader Co-culture, thin rods, spirillaD-i Beige, entire, convex, mucoid No growthE-1 White, entire, convex, mucoid No growthE-2 Beige, wrinkled, mucoid No growth

Pour Plates

Pour plate isolations resulted is isolation of 4 different morphologies from the lobster Cabdomen samples. Plates showed growth within 4 days. Of particular interest were small yellowcolonies which were ovoid when grown within the agar plate, but were spreaders upon thesurface. These colonies initially contained what appeared to be “red sparkles” within the colony.As the cells grew across the surface of the plate, these “sparkles” went with them. As the culturesaged, the red turned to green and then to blue. Different light angles and intensities did not alterthe position or color of the inclusions. Examination of the cells microscopically did not indicateany inclusion bodies or suggest what might be the source for this “optical property”. Fluorescentexamination did not reveal any further insight (Figure 23). Further attempts to culture this isolatewere made, however the morphology was distinctly spreader form, no singular colonies wereretained and all further transfers resulted in only greenlblue “sparkles”. After initial growth, nored “sparkles” were observed, indicating that something may have been lacking in the media orwas diluted out from the initial sample. Additionally, the growth rate of the organism wasexceedingly slow and visual signs of growth did not occur until approx. 10 days. Consideringthat the organism may be microaerophilic, additional pour plates were made from colonyscrapings in an attempt to hasten the growth of the organism. While the organisms grew quickerin the new plates, the colonies were much smaller and remained within the agar, never achievingtheir spreader morphology. This isolate did not ever exhibit signs of chitin degradation.

After noting the preference for microaerophilic growth of the yellow bacteria, a series ofmotility stabs were done to determine level of motility, preference for oxygen concentrations andwhether or not buffer inclusion would improve growth rate. However, there was no detectablegrowth in any of the tubes after a 7 day period. There was insufficient time to continue theobservations. This organism along with several others will continued to be studied at a differentlocation.

Anaerobic tubes developed small black pinpoint colonies with small circular areas ofchitin clearing. Colonies appeared to be entire and convex, growing in the upper 2/3 of the tubes.These colonies only appeared within the last three days of the course and further characterizationwas not possible and will have to continue at another time.

15

MOLECULAR ANALYSIS OF MICROBIAL COMMUNITIES

DNA Extraction

Extraction 1 (control lobster) did not result in any detectable amount of DNA. This waslikely a result of either insufficient quantities of shell removed during scraping or that due to theintact condition of the shell, sterile washes removed any attached bacteria. All other extractsresulted with high molecular weight DNA of sufficient quantity and quality for PCR.

PCR reactions

Amplification of bacterial 16S rDNA was achieved in all eight lobster samples, with thegreatest amount of product produced for sample 7 (white spot, under-pereiopod). Amplificationof archaeal 1 6S rDNA resulted in four amplifications for samples 2 through 5 (Table 2). PCRproduct was 1400 bp in size as expected and of sufficient quantity for cloning.

Cloning

Samples 2, 3, 5, and 6 (Table 2) had amplification with the bacterial primers,additionally, samples 2, 3, 4, and 5, also had amplification with archaeal primers. Each of thesesamples was cloned from their prospective PCR product. E. coli clones were incubated for 24 hon LB media prior to selection for reamplification. White colonies (4 from each of 8 positivesamples) were selected for colony pick PCR with M13 primers. Additionally, each white colonyselected for PCR, as well as up to 10 other positive clones, were transferred to fresh media for asecond screening. These transfers were allowed to incubate for 24 h and checked for correctcolony morphology. PCR was limited to only four colonies from each sample due to time andsupply restrictions of the lab. Following amplification, products were assessed for presence ofcorrect fragment insert size. Each of the 32 amplifications had surplus bands in the products,indicating possible mis-priming, or non-optimal conditions for the amplification. Additionally,several of the clones picked for analysis reverted to negative morphology upon completion ofsecond screening. Each sample did have the correct size insert, however due to the presence ofother bands samples were not useful for performing ARDRA screening or subsequentsequencing analysis. Numerous clones which were transferred, but not picked for analysisremained positive for insert selection, allowing the possibility for further investigation at a futurepoint in time.

CONFOCAL MICROSCOPIC EXAMINATION OF MOLTED SHELL

Pieces examined using different fluorescent stains and wavelength of light wereenlightening as to the formation of bioflims on the carapace regions. The DAPI stained shell wasexamined using 800 nm 2 and resulted in visualization of bacteria in clumps along the borderedges of degraded shell and suspended in what appeared a type of mucoid or polysaccharideglobules. Pits within the shell were easily visualized as well. SYBR Green stained shell wasmuch more detailed and individual organisms were easily viewed using 488 nm 2. Clarity anddepth of field were much improved over the other stain types. Additionally, the shell had somebackground illumination which allowed much clearer pictures of the exact locations of the

16

organisms which were attached to the degraded area. Images were saved on CDs for laterexamination, including 3-D animation which allowed better depth perception for viewing areasof colonization.

Conclusions

Using the dilution-to-extinction technique, organisms which were likely numericallyrelevant in the lobster wound were enriched in liquid media. Four pure cultures were isolated.The first isolate was able to use chitin as the sole carbon and nitrogen source. The second isolatewas able to use peptone (partially digested proteins) as the sole carbon and nitrogen source. Thethird isolate secreted lipase and was able to use Tween® 80 as the sole carbon source. The fourthisolate was able to grow in a mixture of chitin, peptone, and Tween® 80. The next step will beto sequence the pure cultures and compare them to the types of organisms that have previouslybeen associated with shell disease.

The pure cultures were applied to a healthy, sterile moltshell to determine if shell diseasecould be induced. After a four-day incubation period, black spots appeared on the test shell. Theblack spots may be due to the action of the lipase-secreting isolate, which could break down thelipids present in the outer layer (epicuticle) of the lobster shell. This shell will continue to bemonitored to determine if the black spots grow into the typical lesions of shell disease. If shelldisease develops, it would then be informative to repeat the experiment using one pure cultureper fresh shell to see if a single isolate can cause shell disease. It is possible that a consortium ofmicroorganisms is required for shell disease; for example, in the absence of a shell wound, alipase-secreting organism may be required to break down the epicuticle which would then allowa chitinoclastic microorganism to reach the chitinous shell layers.

T-RFLP was used in an attempt to compare the bacterial communities present in theseawater column, “cigarette burn” shell disease, and “white spot” shell disease. It is of especialinterest to see if the microbial communities present in the different manifestations of the diseaseare different. Differences in the microbial community may be able to account for the differentappearance of the disease. T-RFLP analysis did not yield useful information, likely because theterminal fragments were very short and quickly traversed the gel. The analysis should berepeated by using a different restriction enzyme so that significant comparisons can be madebetween samples.

Confocal microscopy was used to visualize the biofilms that grew on lobster shell thatwas suspended in the scum layer of the tank housing lobsters B and C. Rods were the dominantmorphotype in the biofllm which was expected based on the dominance of rods in the liquidenrichments of wound microorganisms.

Cultivation of chitinoclastic bacteria is possible from lobsters exhibiting shell disease.The isolates obtained are distinctly different morphologically between the types of diseaseetiology and control specimens. Chitin degrading isolates were obtained from different types ofmedia and culture conditions. Several organisms of interest and of unique properties wereisolated and will continued to be studied to determine their role in the aggravation of the disease.

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Molecular analysis of the microbial communities via cloning was not successful in thisone attempt to characterize the species present from communal DNA. Further analysis of theclones will permit a better characterization of the populations present. Additionally, otheranalysis may be possible from DNA obtained which may give other suggestions for direction offuture research. The isolates obtained will need to be characterized via 16S rDNA analysis at afuture time. Given the large variety of organisms present within the wounds (as determinedmicroscopically), the identification of these species is bound to be an interesting endeavor. Thetime period available within this course is insufficient for adequate examination of a complexsystem such as this one. The microbial communities are in dynamic flux with the lobster andwater conditions, which prevents easily accessible answers as to whom the individual species arewhich are involved.

References

Bayer, R., D. Prince, C. Waltz et al. 1989. Scanning electron microscopy and X-ray analysis ofshell disease lesion in the American lobster. I Shellfish Res. 8:48 1-482.

Baross, J. A. and P. A. Tester. 1975. Incidence and etiology of exoskeleton erosion in the spidercrab Chionocetes tanneri Rathbun (Brachyura: Majidae). Paper presented at AIBS meetings(Society of Invertebrate Pathologists), Aug. 1975, Oregon State University, Corvallis, Oregon.

Bergey’s Manual Vol. 3 Non-photosynthetic, non-fruiting, gliding bacteria.

Dennel, R. 1960. Integument and Exoskeleton. In The Physiology of Crustacea. T. H.Waterman, ed. Vol. 1. pp. 449-472. Academic Press, New York.

Enos, E. 2000. Personal communication. Marine Biological Laboratory, Marine ResourceCenter, Woods Hole, MA.

Estrella, B. 1991. Shell disease in American lobster (Hornarus americanus, H. Mime Edwards1937) from Massachusetts coastal waters with considerations for standardizing sampling. IShellfish Res. 10:483-48 8.

Fisher, W. S., T. R. Rosemark, and R. A. Shleser. 1976. Toxicity of malachite green to culturedAmerican lobster larvae. Aquaculture. 8:15 1-156.

Fisher, W. S., E. H. Nilson, J. F. Steenbergen, and D. V. Lightner. 1978. Microbial diseases ofcultured lobsters: A review. Aquaculture. 14:115-140.

Floreto, E. A. T., D. L. Prince, P. B. Brown, R. C. Bayer. 2000. The biochemical profiles ofshell-diseased American lobsters, Homarus americanus Milne Edwards. Aquaculture 188: 247-262.

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Gerhardt, P. (ed.), R. G. E. Murray, W. A. Wood, and N. R. Krieg (eds.) 1994. Lipase Activity,25.1.44.2. Methods for General and Molecular Bacteriology. American Society forMicrobiology, Washington, D.C.

Getchell, R. G., 1989. Bacterial shell disease in crustaceans: A review. .1 Shellfish Res. 8:1-6.

Jackson, C. R., E. E. Roden, and P. F. Churchill. 1998. Changes in Bacterial SpeciesComposition in Enrichment Cultures with Various Dilutions of Inoculum as Monitored byDenaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, 64 (12):5046-5048.

Malloy, 5. 1978. Bacteria induced shell disease of lobsters (Homarus americanus). I Wildl. Dis.14:2-10.

Prince, D., R. Bayer, M. Gallagher, and M. Subramanyam. 1995. Reduction of shell disease withan experimental diet in a Nova Scotian lobster pound. I Shellfish Res. 14:205-207.

Prince, D. 1997. Studies on the etiology and pathogenesis of shell disease in the Americanlobster, (Homarus americanus). PhD. Thesis, University of Maine, ME USA.

Stewart, J. E. 1980. Diseases. In The Biology and Management ofLobsters. J. S. Cobb and B.F. Phillips, eds. Vol. 1. pp. 301-342. Academic Press, New York.

Syslo, M., 2000. Personal communication. Vineyard Lobster Hatchery. Martha’s Vineyard, MA

Unestam, T., and D. W. Weiss. 1970. The host-parasite relationship between freshwatercrayfish and the crayfish disease fungus Aphanomyces astaci: Responses to infection by asusceptible and a resistant species. I General Microbiology. 60: 77-90.

Acknowledgements

The authors would like to thank Ed Enos for providing information regarding lobster careand procurement, Michael Syslo and Dr. Rainer Voigt for providing diseased, and Dr. RoxannaSmolowitz for helpful discussions on etiology of the disease. Additionally, the faculty and staffwho provided advice and instruction during the course, are gratefully thanked. Special thanksgoes to Alfred Spormann for his assistance with confocal microscopy, Jared Leadbetter formedium design discussions, and Amy Schaefer for knowing where everything is! Finally,recognition must be given to the lobsters who gave their lives for science. May LobstersAlfreda, Bernice, and Della rest in peace. Lobsters Caroline and Emily were placed in a witnessprotection program and their whereabouts are unknown at this time.

.19

__

- i&- •1 —- -,

S -

t f - —

I’ r

Tail

r

Abdomen

r

3rd5th

r

Carapace

1st Pereiopod

Pereiopod

Figure 1. Anatomy of American lobster with parts susceptible to shell disease annotated.

20

Figure2. Close-up view of lobster carapace. Panel A represents and normal healthy intactcarapace. Panel B represents an example of advanced shell disease on the carapace.

21

Figure 3. Two forms of shell disease commonly exhibited on Pt pereiopods of two different

lobsters. Panel A is classical brown spot degradation. Panel B is white spot degradation

commonly found in captive lobsters. Arrows indicate points of disease.

22

.

.

.

Figure 4. Healthy Shell (Abdomen, Lobster E)

23

Figure 5. “Cigarette Burn” Shell Disease (Carapace, Lobster A)

Figure 6. “White Spot” Shell Disease (1st Perelopod, Lobster D)

24

Figure 7. Phase Contrast Micrograph (40x) of Chitin Liquid Enrichment

(1:100,000 dilution)

Figure 8. Phase Contrast Micrograph (40x) of Peptone Liquid Enrichment

(1:1,000,000 dilution)

.

.

.25

Figure 9. Phase Contrast Micrograph (40x) of Tween 80 Liquid Enrichment

(1:1,000,000 dilution)

Figure 10. Phase Contrast Micrograph (40x) of Mixture Liquid Enrichment

(1:100,000 dilution)

26

Figure 11. Phase Contrast Micrograph (lOOx) of Pure Culture

Isolated on Chitin Agar Medium

Figure 12. Phase Constrast Micrograph (lOOx) of Pure Culture

Isolated on Peptone Agar Medium

Figure 13. Phase Contrast Micrograph of Pure Culture

Isolated on Tween® 80 Agar Medium

.

.

.27

Figure 14. Phase Contrast Micrograph (100 x) of Pure Culture

Isolated on Mixture (Chitin, Peptone, Tween® 80) Agar Medium

Figure 15. Colony Surrounded by Calcium Soap Precipitate

Growing on Agar Plate Supplemented with Tween® 80

28

I

.

L

Figure 16. Control Moltshell After Four-Day Incubation

I

Figure 17. Test Moltshell After Four-Day Incubation

29

Figure 18. T-RFLP Banding Pattern

30

Figure 19. Confocal Micrograph of Bioflim on Lobster Moltshell (100X)

Figure 20. Confocal Micrograph of Spirilla in Bioflim on Moltshell (100X)

G

.

.31

Figure 21. Chitin degrading bacteria isolated from lobsters grown on chitin overlay media.

Plate growth and chitin removal after two weeks of incubation. Top image half plate view and

lower image close-up of streak plate colonies, arrow indicates area of chitin removal.

32

A

. %

Figure 22. Images of microorganism isolated from lobster C abdomen sample, whichdemonstrated chitin degrading capabilities. Potentially of the genus Saprospira based uponmorphological and growth characteristics. Panel A is 40X magnification, panel B is IOOX

magnification indicating dark inclusion bodies and panel C is IOOX indicating spheroplast likecells in an older culture.

/ ‘r1 .

¶ ‘- ../I

I I

Ili ) ‘i ./ ‘

/ : ; -

‘A \ . — \---:: \ L

Figure 23. Images of microorganisms from the yellow/sparkly colonies isolated from lobster Ccarapace. Panel A shows cells in phase contrast (lOOx) and panel B is same frame usingepifluorescence staining. Cells were stained with acridine orange and viewed through a LuciferYellow filter.

33