**MAMALA BAY STUDY**

**COLIPHAGE AND INDIGENOUS PHAGE IN MAMALA BAY**

**PROJECT MB-7**

Principal Investigators:

**John H. Paul**

**Department of Marine Science**

**University of South Florida**

**140 Seventh Ave. S.**

**St. Petersburg, FL 33701**

**Joan B. Rose**

**Department of Marine Science**

**University of South Florida**

**140 Seventh Ave. S.**

**St. Petersburg, FL 33701**

**JUNE 15, 1995**

# 1 EXECUTIVE SUMMARY

A study of the distribution of bacterial viruses (bacteriophages) in
Mamala Bay was undertaken in conjunction with the Microbiological group,
MB-7. Coliphages, or viruses which infect the bacterium *E. coli* and
are indicators of fecal pollution, were enumerated along with total
viral direct counts, and phage 16-like vibriophages. The latter are a
type of marine bacteriophage that we have found abundant in the coastal
waters of Florida. Samples were collected approximately quarterly over a
thirteen month period. Coliphage were found during each quarterly
sampling along an offshore transect to the Sand Island Outfall. High
concentrations were also found in three of four samplings in the Manoa
Stream near the University of Hawaii campus. During the storm event
sampling in February 1994, the nonpoint coastal stations (Pearl Harbor,
Ala Wai Canal, and Hanauma Bay) had high levels of coliphages, but much
lower or none when sampled during dry weather. Coliphages were absent at
all samplings at Waikiki Beach and at the control station off Diamond
Head. Viral direct counts were highest in sewage and in the Manoa Stream
samples (10^11^ and 10^10^, respectively). Eutrophic coastal stations
(Pearl Harbor, Ke\'ehi Lagoon, Ala Moana Beach, and Ala Wai canal)
averaged 10^9^/L. Offshore stations ranged from 9x10^7^ to 1x10^9^
viruses/L, values similar to those for other marine environments. Phage
16-like vibriophages were found mainly in eutrophic coastal environments
(Ala Wai Canal, Pearl Harbor, and Ke\'ehi Lagoon) and in the Sand Island
Transect stations D1 and D2. The greatest abundance was found during the
storm event (February) sampling.

Collectively these results indicate that the fecal indicator phages
(coliphages) were abundant in the Sand Island transect during all
quarterly samplings, were prevalent in the coastal stations during storm
events, and were never found on Waikiki Beach and offshore control
sites. Thus, the major input of indicators into the coastal zone and
beaches seems to be from land runoff entering via non-point sources.
Thus, better treatment of stormwater is recommended.

# Table of Contents

1 EXECUTIVE SUMMARY ii

TABLE OF CONTENTS iv

LIST OF FIGURES v

LIST OF TABLES v

2 INTRODUCTION

2.1 Scope of Work

2.2 Objectives

2.3 Project Organization

3 METHODS

3.1 Task Summary

3.2 Task Methodology

4 RESULTS

5 CONCLUSIONS

6 RECOMMENDATIONS

6.1 Recommended Actions

6.2 Future Studies

7 REFERENCES

8 APPENDICES

# List of Figures

Fig. 1. Coliphage-Means of All Seasonal Samplings

Fig. 2. Coliphage abundance for stations sampled in October 1993

Fig. 3. Coliphage abundance for stations sampled in February 1994

Fig. 4. Coliphage abundance for stations sampled in June 1994

Fig. 5. Coliphage abundance for stations sampled in November 1994

Fig. 6. Viral Direct Counts-Means of All Seasonal Samplings

Fig. 7. Viral Direct Counts for stations sampled in October 1993

Fig. 8. Viral Direct Counts for stations sampled in February 1994

Fig. 9. Viral Direct Counts for stations sampled in June 1994

Fig. 10. Viral Direct Counts for stations sampled in November 1994

Fig. 11. Vibriophage-Means of All Seasonal Samplings

Fig. 12. Vibriophage abundance for stations sampled in February 1994

Fig. 13. Vibriophage abundance for stations sampled in June 1994

Fig. 14. CLUSTAL Analysis of Cryptic Gene Sequence from Related
Vibriophages

# List of Tables

Table 1. Description of Stations Sampled

Table 2. Results of RT-PCR detection of MS-2 coliphage: Comparison to
plaque titers

#  {#section .unnumbered}

# 2 INTRODUCTION

## 2.1 Scope of Work

Mamala Bay is a complex system that encompasses the entire south shore
of the island of Oahu. This system includes estuarine environments
(Pearl Harbor, Ke\'ehi Lagoon, Honolulu Harbor), beaches used for
tourism (Waikiki, Ala Moana Park), major shipping and port activity,
numerous freshwater and stormwater inputs, and wastewater effluent
inputs (Honouliuli outfall, Sand Island outfall, and Pearl Harbor
outfall). The Mamala Bay Study Commission has brought together a suite
of investigators studying the physical, biological, and public health
aspects of this system yielding an integrated coastal management plan
for Mamala Bay. Our project is an important component of this total
picture, the coliphage and indigenous phage in Mamala Bay. We have
worked closely with the other microbiologists in the MB-7 program to
coordinate sampling and share results. Our data also gives a complete
picture of the bacteriophage distribution in this system, and can
\"stand alone\" as a separate publication from this study.

[Coliphage as indicators of water quality.]{.underline} Coliphage
abundance has become an acceptable indicator of water quality. Coliphage
are viruses of the bacterium *Escherichia coli*, a fecal coliform.
Because of viral stability compared to its host, coliphage appear to be
better indicators of marine water quality and are in fact found to be
more persistent in the environment (Simkova et al., 1981). Recent
microbiological reports state that coliphage are the best indicators, to
date, of human enteric viruses in polluted waters (Sobsey, 1989) and
based on the isolation of phage at various bathing beaches, should be
used for continuing evaluation of recreational waters (Palmateer et al.,
1991). These bacteriophages have not yet been detected in unpolluted
waters (IAWPRC Study on Health Related Water Microbiology, 1991). In
addition, coliphage are easily and inexpensively isolated in the
laboratory. A specific example of a potential indicator coliphage is the
MS2 virus. It is an F-specific RNA phage with an *E. coli* host. Since
MS2 can be found anywhere its bacterial host is found, it is capable of
indicating fecal contamination. MS2 has been recommended for use as a
surrogate for the enteric viruses by the Environmental Protection Agency
(EPA, 1989). More recently the MS2 coliphage has been used as a
microbial tracer in a full-scale wastewater treatment facility to
evaluate disinfection efficacy and transport. The coliphage tracer was
found to represent the fate of human enteric pathogens through the
concurrent monitoring of indigenous enteroviruses in this study (Rose
and Carnahan, 1992). Furthermore, the MS2 virus has been used in
disinfection studies and documented as being highly resistant when
compared to bacterial indicators (Bonde, 1975). Coliphage may therefore
still be present in chlorinated sewage after bacterial indicators cannot
be detected.

The use of these indicator systems has focused on the sanitary quality
of water. There has been no attempt to correlate the occurrence of these
indicators with the level of urbanization and magnitude of human
activities impacting the marine environment in a spatial monitoring
program in the water column.

[The Abundance of Viruses in the Marine Environment.]{.underline}
Viruses are now known to be the most numerically abundant form of life
in the surface waters of our planet. This revelation has come to light
relatively recently, because of the development of methods to enumerate
viruses in water samples directly by transmission electron microscopy,
termed viral direct counts (VDC; Bergh et al., 1989; Proctor and
Fuhrman, 1990; Paul et al., 1991). These viruses are believed to be
primarily bacteriophages, although viruses capable of lysing
phytoplankton are also common in surface waters (Suttle et al., 1990,
Waterbury et al., 1993). The role of viruses in water column
trophodynamics are not completely understood, but it is thought that
they can cause 10-50% of the bacterial mortality (Jiang and Paul, 1995).
In general, the highest viral direct counts are associated with
eutrophic environments, such as estuaries, and polluted lakes (Bergh et
al., 1989; Paul et al., 1991; Boehme et al., 1993). Thus, viral direct
counts can be an indicator of relative trophic status of surface waters,
as bacterial direct counts and chlorophyll [a]{.underline} are often
used. Thus, we felt that it was important to measure these in the Mamala
Bay system.

[Vibriophages.]{.underline} Historically, the abundance of phages in
seawater was determined by plaque titer (Moebus, 1980). The problem with
this approach is that it drastically underestimated the number of
viruses present, because only a few hosts could be used. We have become
interested in the distribution of viruses which infect a marine host we
have isolated termed host #16, tentatively identified as *Vibrio
parahaemolyticus* strain 16. We have isolated phages on this host from
the Gulf of Mexico, the Dry Tortugas, Key Largo, and Tampa Bay. We are
embarking on a study of the genetic diversity of the phages which infect
this host (Paul et al., 1993; Kellogg et al., 1995). As part of this
study, we measured the abundance of such vibriophages in the waters
around Mamala Bay.

## 2.2 Objectives

The study described below was undertaken to understand the distribution
and dynamics of coliphage and indigenous phage in Mamala Bay. These data
will be helpful in assessing the impact of the Sand Island sewage
outfall on the health of Mamala Bay and the users of its marine
resources.

**Objective 1.** To enumerate viruses by in water column samples by
direct counts.

**Objective 2.** To enumerate coliphages by plaque agar overlays.

**Objective 3.** To enumerate vibriophages by plaque titer overlay.

**Objective 4.** Using PCR technology, to detect coliphages and
vibriophages by amplification in select samples.

## 2.3 Project Organization

Dr. John Paul was the principal investigator, in charge of field
operations, viral direct counts, and vibriophage studies. Dr. Joan Rose,
co-PI was in charge of coliphage analysis. A series of graduate students
participated in sampling and sample analysis, including Sunny Jiang,
Chris Kellogg, Pam London, Xingting Xhou, and Jordan Brown.

# 3 Methods

## 3.1 Task Summary

The major tasks were to concentrate samples collected in the quarterly
sampling for viral direct counts, coliphage enumeration, and vibriophage
enumeration. Certain samples were selected for further analysis by PCR.

## 3.2 Task Methodology

[Sampling sites]{.underline}. A description of the sampling sites
appears in Table 1 and the approximate locations can be seen in the data
figures 1-13. Sampling occurred over four quarters in 1993-1994: October
25-29, 1993; February 14-17, 1994; June 20-24, 1994; and November 14-18,
1994. It rained heavily on Oct. 25, and during the whole week of the
February sampling.

[Sampling procedures]{.underline}. Beach and shore-based sampling was
accomplished by immersion of a sterilized 20 l carboy in the water to be
sampled. For offshore samplings, a gasoline driven impeller pump was
used. This was fitted with up to 75 m sample hose (1\" id) for sampling
depths up to 75 m. All samples were returned to the lab and processed
usually within 4 h of collection, but always within 8 h of collection.

[Concentration of Samples for Phage Analysis]{.underline}. 5 liter or 20
liter samples were concentrated by vortex flow filtration using a
Membrex Rotary Biofiltration device (Membrex, Inc, Garfield, NJ)
equipped with a 400 cm2, 100 kd filter (Paul et al., 1991; Jiang et al.,
1992). The systems were run at 1500 RPM and 8-12 PSI, and the final
retentate volume was 45-65 ml. This procedure has been shown to be
70-99% efficient for the concentration of phage and microbial
populations (Paul et al., 1991; Jiang et al., 1992). Sand Island sewage
effluent (S1) samples were not concentrated.

[Coliphage analysis]{.underline}. For coliphage analysis, one and 0.1 ml
samples in triplicate of the retentate or S1 effluent were plated by top
agar overlay using the coliphage hosts *E. coli* ATCC 15597 and/or *E.
coli* Cabelli. Hosts were grown to mid stationary phase on
trypticase-soy broth (TSB; Difco) medium immediately prior to top agar
overlay.

[Vibriophage/indigenous phage isolation.]{.underline} Two marine
bacterial hosts were used for phage isolation in this study: host #16,
tentatively identified as a *Vibrio parahaemolyticus* strain, and HSIC,
an unidentified marine bacterium isolated from Ke\'ehi Lagoon. The
latter was used only in the first two quarterly samplings. Hosts were
grown into log phase on ASWJP medium (Paul, 1982) at room temperature
(24-26°C) with shaking. One ml and 0.1 ml aliquots of the retentate from
each station were assayed for the presence of these phages by top agar
overlay.

[Viral Direct Counts]{.underline}. A portion of the Vortex flow
filtration retentate (usually 12 ml) was fixed with EM grade
glutaraldehyde (Sigma Chemical Co.; 2% final conc.) The sample was
further concentrated by ultracentrifugation at 201,000 xG for 90 min in
a table top ultracentrifuge (Beckman). The sample was then diluted in DI
water to reduce salt content and 1 ml was spotted onto a formvar coated
TEM grid. The grids were air dried and stained with uranyl sulfate. The
grids were examined at 25,000 to 48,000 X with a Hitachi 5500 TEM or a
Hitachi 7100 TEM.

[Amplification of MS-2 coliphage.]{.underline} Primers were designed
based on the MS-2 replicase gene sequence. The \"upstream\" or 5\'
primer sequence (designated #2175) was 5\'-CAA GTT GCA(G) GGA TGC AGC
GCC-3\' and the 3\' primer (#2375) as 3\'-GCC CGA CGG ACA TTC CTC
GG-5\'. The retentates were purified prior to reverse
transcriptase-linked PCR (RT-PCR) by the method of Abbaszadegan et al.
(1993). Briefly, 0.3 ml of the retentate was passed over a mixed bed
column consisting of Chelex 100 and Sephadex 100 in a 1.0 ml disposable
syringe. Five ml of the purifed retentate was used in a RT reaction
containing 1x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 1 mM each
dNTP, and 5 mM MgCl~2~. The reaction was heated in a thermal cycler for
5 min at 100°C, followed by cooling at 4°C. RNasin (9 units), reverse
transcriptase, and 1 mM of 3\' primer. The reaction was incubated for 10
min at 24°C, 42°C for 50 min, 99°C for 5 min, and 4°C for 5 min. The
entire RT reaction (30 ml) was added to a PCR reaction mixture
containing 1x PCR buffer, 0.3 mM each primer (3\' and 5\'), 2.5 mM
MgCl~2~, and 2.5 units Taq Polymerase in a total volume of 100 ml. The
first cycle of amplification consisted of denaturation at 95°C for 2
min, annealing at 50°C for 30 s, and extension at 72°C for 45 s,
followed by 30 cycles of heating at 95°C for 1 min, annealing at 45°C
for 45 s, and extension at 72° for 45 s. The final extension (last
cycle) was at 72°C for 7 min.

[Detection of Vibriophage by amplification]{.underline}. Samples were
further concentrated for PCR by the method of Tsai et al., 1991. Fifteen
ml retentate was concentrated to \~4.0 ml by centrifugation using an
Amicon Centriprep (100 kd filter). The concentrated sample (\~4 ml) was
extracted with an equal volume of Chloroform:isoamyl alcohol,
centrifuged for 5 min at 800 rpm for 5 min. The supernatant was
transferred to an Amicon Centricon and concentrated to 300-500 ml. The
concentrated extract was precipitated in two volumes of ethanol at -20°C
until processing. The ethanol precipitates were collected and washed
with 70% ethanol, dried, and dissolved in 300 μl sterile DI. The samples
were then further purified by passage over mixed bed columns of Chelex
100 and Sephadex G-100 as described by Abbaszadegan et al. (1993). The
eluates were ethanol precipitated and redissolved in DI water.

[Genetic analysis of vibriophages]{.underline}. Representative plaques
from various samplings were selected for further study by genetic
analysis. Plaques were isolated and repurified by top agar overlay. The
DNA was purified by the method of Maniatis et al. (1982) as modified by
Kellogg and Paul (1995). A 488 bp fragment was cloned from these phages
into M13 after PCR, and the fragment sequenced using the Sequenase kit
(US Biochemicals). The sequences were compared to other vibriophage
isolates from the Gulf of Mexico using CLUSTAL and maximum likelihood
analysis.

# 4 Results

[Detection of Coliphage.]{.underline} The overall seasonal average
coliphage data appears in Fig. 1 and seasonal values appear in Figs 2 to
5. The highest concentration of coliphage was found, not surprisingly,
in the Sand Island sewage effluent, ranging from 1.4 to 2.78 x 10^6^/L.
Coliphage were found in every seasonal sampling at certain stations
along the Sand Island transect. The highest concentrations of coliphage
were found consistently in the surface waters of station D1, which was
located shoreward of the sewage outfall D2, Levels of coliphage showed
little variation with season in the Sand Island transect, with the
exception of the November sampling (Fig. 5), when only 3 of 7 samples in
this transect were positive.

The Manoa Stream samples at the University of Hawaii campus contained
high levels of coliphage in three of four quarterly samplings, with none
detected in the February sampling (Fig. 3). Stations located in Ala Wai
canal contained coliphage in two of four samplings (October and
February; Figs. 2 and 3). These sampling times were also characterized
by rain, especially the February sampling (Fig. 3). Stations in Pearl
Harbor were positive for coliphage only in February and June. Hanauma
Bay, Sand Island Beach, and Ala Moana Beach only had one positive each,
and two of these occurred in the February sampling (Fig 3).

No coliphages were ever detected at the Diamond Head Control station at
all depths, nor were any found at the Waikiki Beach samples.

The non-point sources of input into Mamala Bay (i.e. Pearl Harbor, Ala
Wai Canal, Hanauma Bay and Ala Moana Beach) seemed to have the highest
coliphages during the storm event sampling. This is consistent with land
runoff (storm sewers, culverts) into coastal waters. However, this input
source did not apparently influence Waikiki Beach, which had no
coliphages at any sampling. Even though the Sand Island transect
contained coliphage during all samplings, these apparently were not
influencing Waikiki Beach.

[Detection of coliphages by PCR.]{.underline} Table 2 shows the results
of the detection of coliphage by PCR in comparison to plaque titers.
There was agreement in the results obtained by the two methods in 58 of
71 samples (81.6%). For the samples that did not agree, 2 of 71 (2.8%)
were positive by PCR but negative by plaque titer, and the remainder (11
of 71; 15.5%) were negative by PCR but positive by plaque titer. In some
of the latter samples, it may be that there were coliphage that were
present that were not MS-2, for which the primers were designed.

[Viral Direct Counts (VDC)]{.underline}. Fig. 6 shows the means of all
seasonal samplings for viral direct counts, and figures 7 to 11 show the
individual quarterly data. As with the coliphage date, VDC were greatest
in the raw sewage, averaging \>10^11^/L. The freshwater environment
Manoa Stream had the second greatest concentrations, in excess of
10^10^/L. The eutrophic estuarine stations (Pearl Harbor, Ke\'ehi
Lagoon, Ala Moana Beach, and the Ala Wai Canal (AW1) averaged in excess
of 10^9^/L.

At most stations, viral direct counts were lower in June and November
Fig. 9 and 10), two samplings with little or no rain, compared to
October and February, the wetter samplings. We hypothesize that the
general increase in runoff during the latter two samplings resulted in
greater nutrients and production in the coastal waters, resulting in
higher viral titers.

[Vibriophage.]{.underline} The use of the Hawaiian host HSIC resulted in
no plaques from any samples during the first two quarterly samplings. We
therefore discontinued use of this host. Plaques were found only in
samples from nearshore estuarine samples (Ala Wai Canal, Pearl Harbor,
Ke\'ehi Lagoon, and the Sand Island transect stations D1 and D2; Fig.
11). Unlike the other viruses determined in this study, vibriophage were
totally absent from sewage or freshwater samples (Manoa Stream). As with
coliphage in coastal environments, Vibriophage were most abundant during
the rainy (February 94; Fig 12) sampling. The lowest abundance was in
the October 93 sampling, when these phages were only detected in two
samples (data not shown).

[Detection of Vibriophage by PCR]{.underline}. Several samples were
examined for vibriophage detection by PCR. None of these resulted in
positive amplification. It may be that the concentration of phages were
sufficiently low that amplification did not occur, or that interfering
substances inhibited PCR.

[Genetic Analysis of Vibriophages]{.underline}. Figure 14 shows the
result of clustal analysis of the 488 bp cryptic fragment of the
Vibriophage genome. The one Hawaiian isolate thus far sequenced is most
closely related to F16, an isolate from Tampa Bay.

# 5 Conclusions 

Our data clearly shows a localization of coliphage, indicators of fecal
pollution, around the Sand Island Outfall transect. Coliphage were also
abundant in coastal areas such as Ala Wai Canal, Pearl Harbor, Ala Moana
Beach, Ke\'ehi Lagoon, but only during the wet (February) sampling.
Coliphage were not found nearshore or offshore at Waikiki Beach or at
the control station off Diamond Head, E4. From these observations we
conclude that non-point sources have a greater input of coliphage in the
coastal zone than the Sand Island sewage outfall, particularly during
periods of heavy rainfall. These inputs could therefore influence water
quality at the beaches. However, we only measured one indicator of fecal
pollution, and we understand that some pathogenic viruses have been
isolated from Waikiki Beach (K. Reynolds, University of Arizona,
personal communication). Thus, before conclusions can be made as to the
impact of the Sand Island Outfall on the water quality an integrated
coastal management plan must first be devised.

Our measurements of viral direct counts are the first to be made in
Hawaiian waters. The fact that most oceanic measurements were 108/ml
illustrates the oligotrophic nature of the waters around Oahu, and are
consistent for oligotrophic waters in other parts of the world (Boehme
et al., 1993; Berg et al., 1990). The estuarine waters contained values
similar to other coastal waters (Jiang et al, 1995).

The occurrence of Vibriophage (i.e. those capable of forming plaques on
*V. parahaemolyticus* strain 16) in coastal waters has been found by us
in the Key Largo and other environments previously (Paul et al., 1992;
Kellogg et al. 1995). The genetic similarity of these viruses with those
of the Florida waters is surprising (Kellogg et al., 1995). These data
show that there are circumglobal populations of closely related
bacteriophages in the marine environment.

# 6 Recommendations

## 6.1 Recommended Actions

Our study suggests that even though the waters around the Sand Island
Outfall show indications of fecal contamination, this is not influencing
the beaches of Oahu. Rather, nonpoint sources, with input from storm
drains and canals, seem to have a greater input of indicator
microorganisms into the coastal zone. Thus, it seems that better
treatment of the nonpoint sources might be more valuable in maintaining
water quality in the coastal zone. Such treatment might include
construction of retention ponds for storm drain treatment, aeration of
canals, and other technologies available for treatment of nonpoint
sources and stormwater.

## 6.2 Future Studies

A regular monitoring program of the waters of Mamala Bay for fecal
indicators is advised. Such a plan may already be in existence for the
island of Oahu. After the recommendations are carried out for
modification of stormwater and other nonpoint inputs has occurred, a
second seasonal study of the distribution of indicators is recommended,
to include a storm event sampling. Such a study would determine if an
improvement in nonpoint discharge had been made.

# 7 References

Abbaszadegan, M., M.S. Huber, C.P. Gerba, and I.L. Pepper. 1993.
Detection of enteroviruses in groundwater with the polymerase chain
reaction. Applied and Environmental Microbiology. 59:1318-1324.

Bergh, O., K.Y. Borsheim, G. Bratbak, and M. Heldal. 1989. High
abundance of viruses found in aquatic environments. Nature 340: 467-468.

Bonde, G.J. 1975. In: International Symposium on Discharge on Sewage
from Sea Outfalls, Pergamon Press, London.

Boehme, J., M.E. Frischer, S.C. Jiang, C.A. Kellogg, S. Pichard, J.B.
Rose, C. Steinway, and J.H. Paul. 1993. Viruses, bacterioplankton, and
phytoplankton in the southeastern Gulf of Mexico: Distribution and
contribution to oceanic DNA pools. Marine Ecology Progress Series 97:
1-10.

Environmental Protection Agency, 1989. Guidance manual for the surface
water treatment rule, EPA, Washington, D.C.

IAWPRC Study Group on Health Related Water Microbiology. 1991.
Bacteriophages as model viruses in water quality control. Water
Resources. A.H. Havelaar, ed. 25: 529-545.

Jiang, S.C. and J.H. Paul. 1994. Seasonal and diel abundance of viruses
and occurrence of lysogeny/bacteriocinogeny in the marine environment.
Marine Ecology Progress Series 104:163-172.

Jiang, S.C., J.M. Thurmond, S.L. Pichard, and J.H. Paul. 1992.
Concentration of microbial populations from aquatic environments by
vortex flow filtration. Marine Ecology Progress Series 80: 101-107.

Kellogg, C.A., J.B. Rose, S.C. Jiang, J.M. Thurmond, and J.H. Paul.
1995. Genetic diversity of related vibriophages isolated from marine
environments around Florida and Hawaii, USA. Marine Ecology Progress
120:89-98.

Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY.

Moebus, K. 1980. A method for the detection of bacteriophages from ocean
water. Helgolander Meersunters 34:1-14.

Palmateer, G.A., E.M. Dutka, E.M. Jantzen, S.M. Meissner, and M.G.
Sakeellaries. 1991. Coliphage and bacteriophage as indicators of
recreational water quality. Water Resources 25:355-357.

Paul, J.H. 1982. Use of Hoechst dyes 33258 and 33342 for enumeration of
attached and planktonic bacteria. Applied and Environmental Microbiology
43:939-944.

Paul, J.H., S. C. Jiang, and J.B. Rose. 1991. Concentration of viruses
and dissolved DNA from aquatic environments by vortex flow filtration.
Applied and Environmental Microbiology 57: 2197-2204.

Paul, J.H., J.B. Rose, S.C. Jiang, C.A. Kellogg, and Linda Dickson.
1993. Distribution of viral abundance in the reef environment of Key
Largo, Florida. Applied and Environmental Microbiology 59: 718-724.

Proctor, L.M. and J.A. Fuhrman 1990. Viral mortality of marine bacteria
and cyanobacteria. Nature 343: 60-62.

Rose, J.B. and R.P Carnahan. 1992. Pathogen removal by full scale
wastewater treatment. Department of Environmental Regulation, Division
of Wastewater Reuse.

Simkova, A. and J. Cervenka. 1981. Bulletin of the World Health
Organization 59: 611-618.

Sobsey, M.D. 1989. Inactivation of health-related microorganisms in
water by disinfection processes. Water Science and Technology.
21:179-195.

Suttle, C.A., A.M. Chan, and M.T. Cottrell. 1990. Infection of
phytoplankton by viruses and reduction of primary productivity. Nature
387: 467-469.

Tsai, Y.-L., M.O. Sobsey, L.R. Sagaramo, and C.J. Palmer. 1993. Simple
method of concentrating enteroviruses and hepatitis A virus from sewage
and ocean water for rapid detection by reverse transcriptase-polymerase
chain reaction. Applied and Environmental Microbiology 59:3488-3491.

Waterbury, J.B. and F.W. Valois. 1993. Resistance to co-occurring phages
enables marine *Synechococcus* communities to coexist with cyanophages
abundant in seawater. Applied and Environmental Microbiology 59:
3393-3399.

# 8 Appendices

Table 1. Description of Stations Sampled

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Site Station Quarters Sampled

[Designation 10/93 2/94 6/94 1/94]{.underline}

Manoa Stream,

UH Campus MS Y Y Y Y

Ala Wai Canal at

Ala Moana Bridge AW1 Y Y Y Y

Ala Wai Canal Mouth,

at Green Marker #3 AW2 Y Y Y Y

Waikiki Beach near

Police Station W1 Y Y Y Y

Waikiki Beach offshore

21°13.28 N, 157°48.58W W2 Y Y Y Y

Ke\'ehi Lagoon, first station

in Sand Island transect D0 Y Y Y Y

Sand Island Transect,

Inshore Surface Water

21°17.15N, 157°54.35W D1S Y Y Y Y

Sand Island Transect,

Inshore Bottom Water

21°17.15N, 157°54.35W D1B Y Y Y Y

Sand Island Outfall,

Surface Water

21°16.82N, 157°54.43 D2S Y Y Y Y

Sand Island Outfall,

Mid-depth Water

21°16.82N, 157°54.43 D2M N Y Y Y

Table 1. Description of Stations Sampled (cont.)

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Site Station Quarters Sampled

[Designation 10/93 2/94 6/94 1/94]{.underline}

Sand Island Outfall,

Bottom Water

21°16.82N, 157°54.43 D2B Y Y Y Y

Sand Island Transect

Offshore Surface Water

21°16.40N, 157°55.56 D3S Y Y Y Y

Sand Island Transect

Offshore Bottom Water

21°16.40N, 157°55.56 D3B Y Y Y Y

Diamond Head Control

Site, Surface Water

21°13.28N, 157°48.58 E4S Y Y Y Y

Diamond Head Control

Site, Mid-depth Water

21°13.28N, 157°48.58 E4M Y Y Y Y

Diamond Head Control

Site, Bottom Water

21°13.28N, 157°48.58 E4B Y Y Y Y

Upper Pearl Harbor

Waipio Pt. C1 Y Y Y Y

Mid Pearl Harbor

Bishop\'s Pt. C2 Y Y Y Y

Table 1. Description of Stations Sampled (cont.)

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Site Station Quarters Sampled

[Designation 10/93 2/94 6/94 1/94]{.underline}

Mouth of Pearl

Harbor C3 Y Y Y Y

Hanauma Bay HB Y Y Y Y

Ala Moana Beach AM N Y Y Y

Sand Island Effluent S1 Y Y Y Y

Sand Island Beach SB1 N N Y Y

Honouliuli Outfall,

Surface Water

21°17.00N, 158°01.50 HU1 N N Y Y

Barbers Point

Surface Water

21°16.50N, 158°06.40W BP1 N N Y Y

Honolulu Harbor

Surface water

21°17.31, 157°42.27 HH1 N N N Y

Ewa Beach

(Iroquois Beach) EW1 N N N Y

Table 2. Results of RT-PCR detection of MS-2 coliphage: Comparison to
plaque titers

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

October, 1993 February, 1994 June, 1994 December, 1994

STATION (1st quarter) (2nd quarter) (3rd quarter) (4th quarter)

PFU/L^(1)^ RT-PCR^(2)^ PFU/L RT-PCR PFU/L RT-PCR PFU/L RT-PCR

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

AM1 N.D.^(3)^ N.D. 39.3 + \<2.6 - \<2.5 N.D.

AW1 TNTC^(4)^ + 150 - \<2.2 - \<2.9 -

AW2 2.88 + 1.30x10^4^ + \<2.8 - \<3.2 -

BP1 N.D. N.D. N.D. N.D. \<2.9 N.D. \<2.3 N.D.

C1 \<2.7 N.D. 193.5 + 6.3 - \<2.8 -

C2 \<3.2 N.D. 14.1 + \<3.0 - \<3.1 -

C3 \<2.5 N.D. \<2.29 - \<3.5 - \<3.2 -

D1S 19.0 - 1.2x10^3^ + 1.3 x10^3^ + 3.2 +

D1B \<2.75 N.D. 14.7 + 1.1x10^3^ + 2.1 N.D.

D2S \<2.5 - 24.7 - 544 + 10.6 -

D2M N.D. N.D. 77.7 - 957 + \<3.2 N.D.

D2B 377 N.D. 9.6 + 6.62X10^3^ + \<3.0 N.D.

D3S 5.52 - \<2.6 + 36 + \<3.3 -

D3B 12.2 N.D. 9.2 - 5.4 - \<3.2 N.D.

DOS \<2.6 - 4.6 - \<2.8 - \<2.4 -

E4S \<2.5 - \<2.4 - \<2.2 - \<2.9 -

E4M \<2.7 N.D. \<2.3 - \<2.6 - \<3.2 -

E4B \<2.7 N.D. \<2.3 - \<3.6 - \<2.9 -

EW1 N.D. N.D. N.D. N.D. N.D. N.D. \<2.9 N.D.

HB1 \<2.6 N.D. 270.0 + \<3.0 + \<2.8 N.D.

HH1 N.D. N.D. N.D. N.D. N.D. N.D. \<2.7 N.D.

Table 2. Results of RT-PCR detection of MS-2 coliphage: Comparison to
plaque titers (continued)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

October, 1993 February, 1994 June, 1994 December, 1994

STATION (1st quarter) (2nd quarter) (3rd quarter) (4th quarter)

PFU/L^(1)^ RT-PCR^(2)^ PFU/L RT-PCR PFU/L RT-PCR PFU/L RT-PCR

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

HU1 N.D. N.D. N.D. N.D. \<3.5 N.D. \<3.1 N.D.

MS 8.4x10^3^ + \<13.3 + 785 + 800 +

SB1 N.D. N.D. N.D. N.D. 2.67 N.D. \<2.9 N.D.

SI 1.34x10^6^ + 2.4x10^6^ - 2.78x10^6^ + 1.65x10^6^ +

W1 \<1.9 - \<1.9 - \<2.8 - \<3.1 -

W2 \<2.6 - \<2.5 - \<3.0 - \<3.3 -

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

Notes:

\(1\) Plaque-forming units per liter

\(2\) +: detected in polymerase chain reaction. -: not detected in
polymerase chain reaction

\(3\) Not done (4) Too numerous to count

![](media/image1.emf){width="7.070833333333334in"
height="5.0472222222222225in"}

Fig. 1. Coliphage-Means of All Seasonal Samplings

![](media/image2.emf){width="6.973611111111111in"
height="4.977777777777778in"}

Fig. 2. Coliphage abundance for stations sampled in October 1993

![](media/image3.emf){width="7.915972222222222in" height="5.125in"}

Fig. 3. Coliphage abundance for stations sampled in February 1994

![](media/image4.emf){width="7.290972222222222in" height="5.1125in"}

Fig. 4. Coliphage abundance for stations sampled in June 1994

![](media/image5.emf){width="6.788888888888889in"
height="4.854861111111111in"}

Fig. 5. Coliphage abundance for stations sampled in November 1994

![](media/image6.emf){width="7.035416666666666in" height="5.025in"}

Fig. 6. Viral Direct Counts-Means of All Seasonal Samplings

![](media/image7.emf){width="7.317361111111111in"
height="5.129861111111111in"}

Fig. 7. Viral Direct Counts for stations sampled in October 1993

![](media/image8.emf){width="6.471527777777778in"
height="4.691666666666666in"}

Fig. 8. Viral Direct Counts for stations sampled in February 1994

![](media/image9.emf){width="6.841666666666667in"
height="4.940277777777778in"}

Fig. 9. Viral Direct Counts for stations sampled in June 1994

![](media/image10.emf){width="6.559722222222222in"
height="4.725694444444445in"}

Fig. 10. Viral Direct Counts for stations sampled in November 1994

![](media/image11.emf){width="5.899305555555555in"
height="4.2868055555555555in"}

Fig. 11. Vibriophage-Means of All Seasonal Samplings

![](media/image12.emf){width="6.322222222222222in"
height="4.593055555555556in"}

Fig. 12. Vibriophage abundance for stations sampled in February 1994

![](media/image13.emf){width="6.990972222222222in"
height="5.059722222222222in"}

Fig. 13. Vibriophage abundance for stations sampled in June 1994

![](media/image14.emf){width="5.75in" height="4.333333333333333in"}

Fig. 14. CLUSTAL Analysis of Cryptic Gene Sequence from Related
Vibriophages

CLUSTAL alignment of Vibriophage strains isolated based on sequence
analysis of a cryptic gene fragment. The phage from Mamala Bay (HAWA-4)
was most closely related to F16, the isolate from Tampa Bay.