Assessment of Microbial Diversity nd Iso].aion of lVlicroor ganisms

34 Pages·2012·1.38 MB·English

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Assessment of Microbial Diversity nd Iso].aion of
lVlicroorganisms 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 jIlli,o,.s at L!rbno..-Chainoa:
Microbia1 Diversity
June July 2000
—
Abstract
Freshwater and marine decapod crustaceans commonly exhibit exoskeleton degradation
which is termed shell disease. The disease is an external infection caused by microorganisms
which 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 recent
and severe occurrences warrant a more extensive investigation. Currently, it is estimated that
80% of lobsters in the Buzzard’s Bay, MA have shell disease. From the summer of 1999 to the
spring of2000, every pound system south ofCape Cod has been severely affected by the disease.
This infestation has resulted in large-scale loss ofprimary productivity (up to 35% ofthe market
value) and large culling from the pounds.
Shell-diseased lobsters have been studied for a number of years, but most studies have
focused upon the effects on lobster physiology, rather than focusing on the bacteria species
causing the infection. The limited literature available concerning isolation and characterization
of bacteria associated with the disease suggest that Vibrio, Aeromonas, and Pseudomonas spp.
are suspect organisms. However, detailed analysis ofthe microbial populations and communities
associated with lobster shell disease had not yet been performed.
The current research project focused upon isolation and characterization of bacterial
species present in wounds of infected lobsters. Enrichment approaches, which focused on the
major components of lobster shell (chitin, protein, and lipid), as well as aerobic and anaerobic
isolation techniques were used to culture bacteria which may be participating in the shell
degradation. Pure cultures were isolated from each tested substrate. klentification and
characterization of the isolates are still under investigation. Additionally, a variety of
microscopic examinations were performed on the diseased shells, colonies, and bacterial isolates.
Confocal microscopy indicated that the deteriorating shells were covered with biofilms of
various types and containing numerous morphologies. Community structure analyses (terminal
restriction fragment length polymorphism and cloning) were attempted to compare the microbial
communities found in the different appearances of the disease (“cigarette burn” versus “white
spot”) 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 ofthe cultures isolated from lobster wounds, it was attempted to infect a
healthy shell. Preliminary evidence suggests that this bacterial mixture caused the appearance of
black spots on the shell, but the experiment will continue to be monitored. It is undetermined at
this time whether a single isolate can cause shell disease or whether a consortium oforganisms is
required.
Introduction
Freshwater and marine decapod crustaceans commonly exhibit exoskeleton degradation
which was termed shell disease by Hess in 1937. This is also referred to as rust disease or brown
spot. The disease can take three forms: gross anatomy changes due to carapace degradation
(lesions, ulcers and erosion), attachment ofthe outer exoskeleton to the inner soft body (thereby
preventing removal ofthe shell during molting), and small soft patches where chitin and calcium
are degraded locally, all ofwhich result in an aesthetically displeasing, often weakened, animal.
The blackening ofthe damaged exoskeleton is due to melanin production, which is an attempt to
contain the damage through clotting (Unestam and Weiss, 1970). Unfortunately, melanization
can cause problems during ecdysis (process of molting). Aforementioned, the moltshell may
adhere to the new epicuticle, making the lobster unable to completely shed its moltshell.
The disease is an external infection caused by microorganisms which attack the
components of the lobster shell which include chitin, proteins, and lipids. However, infection
and degradation are not found equally on different parts ofthe lobster anatomy. Estrella (1991)
noted that shell disease most often occurred on the first pereiopods (claws) followed by the
abdomen, carapace, and legs. Estrella (1991) suggested that the high incidence of shell disease
on 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 most
recent and severe occurrences warrant a more extensive investigation. In 1991, 51% ofBuzzards
Bay lobsters had shell disease (Estrella, 1991). Currently, it is estimated that 80% of lobster in
the Bay have shell disease (Syslo, 2000). From the summer of 1999 to the spring of2000, every
pound system south of Cape Cod has been severely affected by the disease, particularly the
difficult molting variety (Enos, 2000). This infestation has resulted in large-scale loss ofprimary
productivity (up to 35% ofthe market value) and large culling from the pounds. Shell-diseased
lobsters can be sold commercially, but they command a reduced price, and are usually used for
canned meat. However, Floreto et at. (2000) determined that the nutritional value ofmeat from a
diseased lobster is similarto that from a healthy lobster, in terms ofessential 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 the
epicuticle are three chitinous layers which are called the exocuticle, calcified endocuticle, and
the non-calcified endocuticle. The epicuticle can be damaged through mechanical abrasion or
enzymatic attack (proteases or lipases). If the epicuticle is repaired in a timely fashion, the
chitinous layers are not subject to attack by chitinoclastic bacteria. Stewart (1980) noted several
factors that could prevent or reduce the efficiency ofepicuticle repair such as poor diet, repeated
abrasion (dredging, fighting, crowded impoundments), and enzymatic attack. It has been
suggested that lipolytic bacteria may degrade the epicuticle, thereby providing an entry for the
chitinoclastic bacteria to reach the chitinous layers (Baross and Tester, 1975).
There are a number offactors that affect molting frequency and therefore the incidence of
shell disease: size, sex, and ovigerous condition of the lobster. Larger lobsters molt less
frequently. 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 have
longer contact times with the causative agent(s) ofshell disease. Thus, ovigerous female lobsters
show the greatest incidence ofshell disease.
Shell disease is contagious when lobsters share a seawater habitat and have repeated
physical contact (Fisher et al., 1978). Shell disease may be associated with municipal and
industrial pollution which cause high organic loading. For instance, Estrella (1991) notes that
polychiorinated biphenyls, heavy metals, and hydrocarbons are prevalent in Buzzards Bay where
the incidence of shell disease is quite high. Thus, pollutant minimization may help to decrease
disease occurrence in the wild. However, more specific measures may be taken to reduce the
incidence ofshell 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 for
eight minutes every other day during the larval period. Getchell (1989) notes that impoundment
hygiene may be improved by removing wastes and disinfecting influent water through ultraviolet
irradiation. Since chitinoclastic bacteria may reach the chitinous shell layers through a wound, it
is also important to reduce wounding. Reducing the number of lobsters per volume, decreasing
the impoundment time, and proper diet can decrease lobster wounding (Getchell, 1989).
Previous research on shell disease has been mainly limited to observations focused on the
lobster physiology (Prince, 1995; Floreto et al., 2000). Fisher et al. (1978) reviewed the six
known microbial diseases of cultured lobsters, which include: shell disease, Gaffkemia,
microbial epibiont, Lagenidium, Haliphthoros and Fusariu,n diseases. The chitinoclastic
microorganisms of shell disease do not penetrate into soft tissues but may provide a portal for
secondary invaders. While most investigations have used adult lobsters, death is more frequent
in larval stages, presumably due to thinner exoskeletons (Fisher et al., 1976). Microbiological
observations have been limited to mainly microscopic examinations using light microscopes, x
ray analysis, and scanning electron microscopy (Bayer, 1989). Literature concerning isolation
and characterization of chitinoclastic bacteria have suggested that Vibrio, Aeromonas, and
Pseudomonas spp. are suspect organisms (Getchell, 1989; Malloy, 1978; Prince, 1997).
However, detailed analysis ofthe microbial populations and communities associated with lobster
shell disease has not yet been performed. With the advent of molecular techniques enabling
more detailed examinations, it is paramount to the lobster industry to investigate not only the
causative agents, but also the potential opportunistic colonizers which may exacerbate the
disease. Additionally, a more detailed identification ofthe pathogenic organisms involved may
lead 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 ofthis
project 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, or
lipids.
• Isolate and characterize pure cultures from mixed community obtained from wounds.
• Determine ifpure cultures oforganisms isolated from lobster wounds can be used to infect a
healthy shell.
• Compare microbial communities present in the seawater column and on a healthy lobster
shell to the microbial communities present in the “cigarette burn” and “white spot”
appearances ofshell disease.
• Isolate DNA and use clone libraries to assess the identities oforganisms associated with shell
disease 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 the
Lobster Hatchery on Martha’s Vineyard. The healthy lobster was obtained from Vineyard
Sound, and the three diseased lobsters were obtained from about eight miles south ofNo Man’s
Island. The diseased lobsters had shell disease that affected the pereiopods, carapace, and
abdomen; this disease manifested itself in the appearance of cigarette burns. Another diseased
female lobster was obtained from Dr. Rainer Voigt of the Marine Biological Laboratory. This
lobster had many white patches, especially around the first pereiopods (claws). A summary of
lobster information is shown in Table 1.
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Table 1. Summary ofLobster Information .
Lobster ID Source Disease Appearance Approx. age (yrs)
A 8 mi. south ofNo Man’s Island Cigarette burns 7
B 8 mi. south ofNo Man’s Island Cigarette burns 7
C 8 mi. south ofNo Man’s Island Cigarette burns 7
D New Bedford, MA pound White patches 8
E Vineyard Sound Healthy control 7
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Housing and Maintenance
The lobsters were housed in 54 x 35 x 35 cm tanks. Each lobster was placed in a separate
tank, except Lobsters B and C shared a tank. Fresh seawater (22 °C) was continuously pumped
through the tanks at a rate 5.3 L/min. The lobsters were fed once or twice a week with fresh or
frozen squid tentacles from the Marine Resource Center.
SAMPLING FROM THE LOBSTERS
Lobsters were removed from the tanks, rinsed thoroughly with sterile seawater, and dried
with Kimwipes® prior to sampling. Samples were collected from the lobsters by swabbing and
scraping the shell with either a sterile swab or scalpel.
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LIQUID ENRICHMENT MEDIA
Synthetic Seawater Media
Four synthetic seawater media were designed for aerobic liquid enrichments: ball-milled
chitin (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 the
nitrogen source, and a medium containing a mixture of chitin, peptone, and Tween® 80. The
medium contained (per liter ofmedium): 20 g NaCI, 3 g M2•206gHC,l 0.15 g C222OaHC,1 0.2
g K2P4,OH 0.5 g KC1, 1 mM N2S4,Oa 4 g ofball-milled chitin OR 4 mL Tween® 80 OR 4 g of
peptone OR all three, 1 mL l000x trace elements solution, 1 mL 1000x 12-vitamins solution, 1
mL vitamin B12 solution, 5 mM Hepes buffer, and 2 mM N3.aHCO The Tween® 80 medium
also contained 0.5 mM N1-I The pH of all media was adjusted to 8 using 10 N NaOH. The
trace elements solution consisted of (per liter): 5200 mg EDTA adjusted to pH 6.0 with NaOH,
2100 mg F4’207eSH,O 30 mg H3B3,O 100 mg M242OnHC,I 190 mg C2’2O6oCH,1 24 mg
N2•2O6iCH,1 2 mg C2•202uHC,l 144 mg Z472OnHSO, 36 mg N2M4•2O2oHaO, 25 mg sodium
vanadate, 6 mg N2S352OeHOa, 8 mg N2W422OHOa. The l000x 12-vitamins solution consisted
of (per 100 mL): 100 mL of 10 mM phosphate buffer (pH 7.2), 10 mg riboflavin, 100 mg
thiamine•HC1, 100 ing L-ascorbic acid, 100 mg D-Ca-pantothenate, 100 mg folic acid, 100 mg
niacinamide, 100 mg nicotinic acid, 100 mg 4-aminobenzoic acid, 100 mg pyridoxineHCl, 100
rng lipoic acid, 100 mg NAD, and 100 mg thiamine pyrophosphate. The l000x vitamin B12
solution consisted of 100 mL DDW and 100 mg ofcyanocobalamin.
The carapace ofLobster B was scraped and a portion ofthe scrapings was placed into 1.0
mL ofeach ofthe four media. Medium (4.5 mLs) was added to sterile 18x150-mm tubes which
were capped with sterile Morton closures. Twenty tubes were prepared so that a ten-dilution
tube experiment could be run in duplicate. 0.5 mL ofthe stock inoculum was transferred into the
first tube, and 1:10 dilutions were made with the remaining nine tubes. The liquid enrichments
were incubated aerobically at room temperature on a shaker table. Terminal dilutions still
showing 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 ofwashed agar was added per liter ofmedium. These plates were inoculated from the
liquid enrichments, using the highest dilution that still showed turbidity. 0.1 rnL was taken from
the terminal dilution, and 1:10 dilutions were performed into sterile 2 mL eppendorf tubes
containing the appropriate liquid medium. 0.1 mLs from the 1:100,000 and 1:1,000,000 tubes
were spread onto the agar plates and incubated aerobically at room temperature. Isolated
colonies were streaked twice for purity.
Aerobic SeawaterMedia to Isolate Chitin-Degraders
Basal seawater medium contained I L freshly collected seawater amended with 15 g
washed agar and was autoclaved. Plates were poured and allowed to harden before the chitin
overlay was added. The chitin layer contained 4.0 g chitin (either ball-milled or unbleached
flaked), 20 g washed agar, 0.70 g K2H4,PO 0.50 g M4•207gSH,O 0.30 g K2P4,OH and 1.0 mL
trace mineral solution per 500 mL distilled deionized water. This was adjusted to pH 8.0 ± 0.2 at
25 °C. After autoclaving, the medium was cooled to 55°C and poured in a thin layer over the
hardened basal media. Plates were allowed to harden and then stored at 5°C until use. Plates
aged for at least 24 hours prior to use to allow a gradient to form between the seawater layer and
distilled deionized water layer.
Fresh swabs from the lobsters were spread in duplicate over the aerobic chitin medium to
form 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 aerobic
media 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 was
boiled to dissolve the agar and then gassed with a mixture of N2/2C0 to reduce the dissolved
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oxygen concentration. The medium was dispensed into baich tubes (10 mL) and sealed prior to
autoclaving. After cooling to 55°C, the tubes were inoculated under gas with samples from
lobsters described below, resealed and gently mixed without introducing bubbles within the
medium. The roll tube method was applied to the sealed tubes to produce a thin solid layer of
chitin medium to the inner surface of the balch tubes. Chitin degradation should be easily
observable via clearing zones within the thin layer formed.
The remaining anaerobic media was used in a series ofpour plates which were inoculated
with material scraped from the diseased lobsters. The pour plates were permitted to go aerobic
on the surface to form an oxygen gradient. The plates were then sealed with parafilm and
incubated at room temperature.
Characterization ofChitin-Degrading Isolates Media
Bacterial isolates obtained from solid media were streaked for purity and transferred
twice. After purification, isolates were inoculated into a variety of motility agar stabs. Basal
stab media contained 0.35 g K2H4,PO 0.25 g M4•207gSH,O 0.15 g K2P4,OH and 0.5 mL trace
mineral solution added to 500 mL of either fresh seawater or distilled deionized water. The
medium was split into 250-mL portions, and each portion received 1 g ball-milled chitin. Each
250-mL portion was mixed and split into 125-mL portions and 1.25 mL 1M MOPS buffer (pH
7.0) was added to one each of the final flasks. Total media was (8) 125-mL aliquots of two
water types, half containing chitin additions and half containing buffer additions. All were
adjusted to pH 8.0 ± 0.2 at 25 °C with 1 M NaOH. Solidifying agent was 0.2 g agarose per flask
which was added prior to autoclaving. After the media cooled to 60°C, 10 mL aliquots were
dispensed aseptically into test tubes which were then vortexed and placed in an ice bath to
harden immediately.
DNA EXTRACTION
Samples 1-8 (Table 2) were collected from each lobster by scraping the outer shell with a
sterile scalpel. Lobsters were removed from each tank, rinsed thoroughly with sterile seawater
and dried with Kimwipes® prior to scraping. The scraped material was collected and added to a
DNA extraction tube (MoBio Inc., Solana Beach, CA) until visually turbid. The accumulated
scum 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-filter
apparatus containing a 0.22-pm filter. The filterwas cut into squares with a sterile razor blade in
a sterile petri dish and then placed in a DNA extraction tube. For DNA from seawater (samples
10-13, Table 2), fresh seawater was filtered through a 125-mm Whatman #1 filter in a Buchner
funnel to remove gross particulates. Then one liter ofthe filtrate was vacuum-filtered through a
47-mm nylon 0.22-tm filter (MSI, Inc., Westboro, MA). As described previously, the filter was
cut into pieces and placed into a DNA extraction tube. This procedure was performed for four
filters, and each filter was placed into a separate DNA extraction vial. DNA was also extracted
from 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 dilution
was 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 quality
was assessed using a 1 % LE agarose gel (lx TBE buffer), run for 35 mm at 150 V, and stained
with GeiStar® (BMA, Rockland, ME).
Table 2. Sample Identification for Isolations and DNA Extractions
Sample Lobste Description DNA PCR
ID r ID Extracted Amplification*
1 B Carapace No
-
2 A Carapace Yes A,B,T
3 B Carapace Yes A, B, T
4 C Carapace Yes A,B,T
5 C 1st pereiopod Yes A, B, T
6 C Abdomen Yes B
7 D White spot, under lstpereiopod Yes B, T
8 D Tan spot on right 1St pereiopod Yes B
9 Tank Filter with 250 mL scum layer Yes B, T
B/C
10 Filter with 1 L seawater Yes T
11 Filter with 1 L seawater Yes T
12 Filter with 1 L seawater Yes T
13 Filter with 1 L seawater Yes
14 B 1:100,000 dilution Yes T
(chitin enrichment)
15 B 1:1,000,000 dilution Yes T
(peptone enrichment)
16 B 1:1,000,000 dilution Yes T
(Tween® 80 enrichment)
17 B 1:100,000 dilution Yes T
(chitin, peptone, Tween® 80
enrichment)
* 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 specific
primers. Bacterial 16S amplification used primers S-D-Bact-0008-F-20 and
19 and archaeal 16S amplification used primers S-D-Arch-2l-F and S-D-Arch-958-R. PCR
reactions contained: 5 jiL lOx reaction buffer; 4 iL 25 mM M2;gCl 2 iL 2.5 mM dNTP mix; 2
iL each forward and reverse primer (30 pMol); 2 jiL sample DNA 125 ng); 0.5 jiL
(‘.-
ArnpliTaq 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 initial
denaturation (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 product
was assessed on 1% LE agarose gel (lx TBE buffer) for 35 mm at 150 V and stained with
GelStar®.
Cloning
PCR products ( 1400 Kb) that were obtained from DNA extracts of lobster shells were
cloned using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) following manufacture’s
protocols. Samples which had sufficient PCR product were cloned. Clones were plated on LB
agar (1 % tryptone, 0.5 % yeast extract, 1 % NaCI, pH 7.0) with 50 jig /mL kanamycin added
after autoclaving. X—gal (40 mg /mL) was added (40 jiL) and spread upon surface after plates
hardened. Both 50 and 80 jiL aliquots of each cloning reaction were plated to ensure adequate
coverage. Clones indicating positive for DNA inserts were selected and tested with PCR
amplification using M13 primers specific for the vector insert region. Products were analyzed on
a 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. PCR
reactions contained: 5 jiL lOx reaction buffer, 4 jiL 25 mM M2,gCI 2 jiL 2.5 mM dNTP mix, 2
1iL each forward and reverse primer (-30 pMol), 1 jiL sample DNA, 0.5 jiL AmpliTaq DNA
polymerase (5U/jiL), and 33.5 jiL sterile distilled deionized water for a total volume of50 jiL. A
25-cycle PCR program was run which consisted of: 95°C initial denaturation (5 mm), 95°C
denaturation 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 TBE
buffer) for 35 mm at 150 V and stained with GeiStar®.
Only samples that showed PCR product were digested. The digestion reaction consisted
ofthe following: 10 jiL PCR product, 2 jiL lOx Buffer A, 2 jiL BSA, 1 4L ofRSA-I, and 5 jiL
ofsterile distilled deionized water. The samples were incubated in a water bath at 37 °C for two
hours. 10 jiL ofthe 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 piece
was sterilized with ethanol and placed into a sterile petri dish. One dish was filled with the
medium containing the mixture of chitin, peptone, and Tween® 80, and an X was scratched on
the shell with a sterile blade. The four pure cultures were swabbed into the X. The last dish was
filled with the medium containing the mixture of chitin, peptone, and Tween® 80, an X was
scratched 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 dissecting
microscope.
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