Microbial Ecology of Intensive Shrimp Production Systems: Islands of Knowledge in a Sea of Ignorance

AquaSG’17 Aquaculture SingaporeSchool of Applied Science, Temasek Polytechnic

October 6, 2017

Shaun M. Moss Oceanic Institute

Hawaii Pacific University Waimanalo, Hawaii USA

Sustainable Culture Technology

Production

System Microbes

Shrimp

Feeds &

Nutrients

Shrimp Diseases

Modified from Lightner 2008

Shrimp Farmacy

  Sci Rep. 2017 Sep 19;7(1):11834. doi: 10.1038/s41598-017-10738-8.

Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei). Qiu L, Chen MM, Wan XY, Li C, Zhang QL, Wang RY, Cheng DY, Dong X, Yang B, Wang XH, Xiang JH, Huang J

Controlled Production Systems

❖ Reduced water exchange / biofloc ponds

❖ Recirculating aquaculture systems (RAS) ➢ Small environmental footprint ➢ Reduced water use ➢ Pathogen exclusion ➢ Year-round production ➢ Inland culture

Amount of Water Used to Produce 1 kg Shrimp

Shrimp SpeciesWater

Exchange / Day (%)

Stocking Density (#/

m2)

Water Use / kg Shrimp (liter/kg) Reference

P. setiferus 25.0 40 64,000 Hopkins et al. (1993)

P. setiferus 2.5 40 9,000 Hopkins et al. (1993)

P. setiferus 0 20 6,000 Hopkins et al. (1993)

P. vannamei <0.5 100 483 Otoshi et al. (2002)

P. vannamei <0.5 200 370 Otoshi et al. (2002)

P. vannamei <0.5 300 352 Otoshi et al. (2002)

P. vannamei 0.4 301 195 Otoshi et al. (2009)

P. vannamei 0.1 408 163 Otoshi et al. (2009)

P. vannamei 0.2 450 98 Samocha (unpub. data)

P. vannamei 2.0 700 219 Moss et al. (2005)

P. vannamei <0.5 828 402 Otoshi et al. (2007)

Commercial Scale Trial at OI

• Shrimp Species/Genetics

SPF L. vannamei from OI’s breeding program

• Stocking Weight 0.52 g

• Stocking Density

828 shrimp/m2 (518 shrimp/m3) • Feed

35% protein-5% squid

Renovation of OI’s Commercial-Scale System

OI Round Pond prior to renovation

OI Round Pond converted into biosecure RAS

(337 m2)

Harvest DataTrial Duration (days) 83

Stocking Density (shrimp/m2) 828 (518)

Stock Weight (g) 0.52

Survival (%) 67.9

Production (kg/m2) 10.3 (6.4)

Harvest Weight (g) 18.3

Growth (g/wk) 1.50

FCR 1.50

Water Use (L/kg) 402

Water Use (%/day) 2.1

Shrimp Condition at Harvest

Physical damage was minimal even at a harvest density of 562 shrimp/m2 (351/m3)

At Harvest After Flushing

The Microbial "Black Box"

The microbial "black box" refers to the fact that researchers are unable to grow many microbes in a laboratory setting, which means they are unknown and mysterious.

Islands of Knowledge

(Who is there? What are they doing? How do they interact?)

1. Microbial structural diversity using microbiology & molecular tools

2. Microbial functional diversity (e.g. pathogens, nitrifiers, denitrifiers,heterotrophs)

3. “Interaction biochemistry“ of bacteria and impacts on systemfunction (e.g. microbe-shrimp competition for feed, flocculation,bacteria as shrimp food)

4. Diversity and stability of gut flora, role of gut flora in shrimp healthand nutrition

Wastewater Treatment Processes

Microbiology Biological Oceanography

BiochemistryAgriculture Sciences

Human Genome Project

QUESTION METHODS

Bacterial abundance & DAPI-staining population dynamics epifluorescence microscopy

quantitative genomics

Bacterial diversity FISH, metagenomics

Population comparisons DGGE, metagenomics

Function & activity Oxygen consumption enzyme activity, metagenomics

Bacterial growth rates BrdU incorporation

Analysis of nutrient fluxes Stable isotopes

Methods used to address mechanistic questions

: (Who is there? What are they doing? How do they interact?)

DAPI staining – broad taxa distinguished by morphology

filamentous bacteria

dinoflagellate

free-living bacteria

Bacterial Abundance in Shrimp Pond Water

Filamentous Bacteria

Bacterial Abundance Over Time (DAPI cell counts)

Cel

ls m

l-1

ELR2 Bacterial abundance

0.00E+00

1.50E+08

3.00E+08

4.50E+08

6.00E+08

day

14 56 63 65

3.32E+076.41E+061.94E+07

4.36E+07

5.19E+08

2.39E+08

3.21E+08

3.13E+07

ELR2 TOTALELR2 Filamentc

Bacterial Diversity Quantitative Analysis Using FISH

Fluorescence in situ hybrdization (FISH) of bacteria in shrimp raceway using 16S rRNA targeted group-specific oligonucleotide probes

Upper panel: Eubacteria (left) and Roseobacter spp. (right) Lower panel: DAPI-stained bacteria of identical microscopic field

Comparison of Microbial Community Strucutre Using DGGE

Comparison of bacterial populations from shrimp guts inhabiting different production systems by denaturing gradient gel electrophoresis (DGGE)

20

HP

LP

Bacterial community differences in gut of P. monodon from ponds showing high productivity and low productivity

• Shrimp sampled from two ponds showing high productivity (HP1 and HP2) and two ponds showing low productivity (LP1 and LP2)(total biomass) (geometric shapes represent individual shrimp and the pond they came from)

• Bacterial community characterised by NGS 16S rRNA sequencing

• Community based on species present and abundance characterised by multi-dimensional scaling

• Two relatively distinct signatures in bacterial community structure can be seen in ponds with high and low productivity

* note geometric shapes represent individual shrimp and the pond they came from)

Slide provided by Dean Jerry, James Cook University

Relative Abundance of Key Bacterial Species

GM GUT Sed Wat GM GUT Sed WatX X X XX X X XX X

X XX X

XX XX XX X

X XX XX XX

Low Productive

Propionigenium

Vibrio

Salinihabitans

Thioalkalispira

Candidatus bacilloplasma

Candidatus aquilunaFerrimonas

Fusibacter

Photobacterium

Genus

ProchlorococcusThiohalophilus

Desulfococcus

High Productive

Desulfobulbus

Data from Dr. Dean Jerry

Bacterial Activity / Growth Rates

Photomicrographs of FISH- and anti-BrdU-HRP stained marine bacteria. A: Eubacteria (probe EUB338); B: Growing cells (BrdU incorporation).

A B

Bacterial Function Ectohydrolases on Biofloc

Functional Diversity • high bacterial abundance • high hydrolase activity

Enzymatic fractionation • high protease, lipase, phosphatase • low glucosidase • bacteria “rob” shrimp of protein & oils in shrimp feed?

Aggregates act as “Enzyme Reactors”

Integration of "Microbial Tools"

R E S EARCH ART I C L E Quantitative role of shrimp fecal bacteria in organic matter fuxes in a recirculating shrimp aquaculture systemChristine Beardsley1, Shaun Moss2, Francesca Malfatti1 and Farooq Azam11Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA; and 2Oceanic Institute, Waimanalo, HI, USA 2011 Federation of European Microbiological Societies

1. What happens to shrimp feed when fed to shrimp? How much feed dissolves into water column? How much feed is consume by shrimp?

2. How much fecal matter is produced by shrimp? Is fecal material an effective substrate for bacteria? What types of bacteria grow on shrimp feces?

3. How do fecal bacteria impact the microbial ecology of the water column?

• Extremely high abundance;derived from shrimp gut

• Explosive growth: from1.9x1010 to 1.7x1011 cells (gdry feces)-1 in 12h

à Generation time: < 4h • Increase from zero to

1.2x1010 cells*l-1 in 12h

• Raceway (8 weeks): 10x asmany bacteria

Bacteria in feces

Bacterial Abundance

0

10

20

30

x 10

10 ce

lls /

g dr

y w

.

Exp. 1 Exp. 2t0 t0 t12ht12h

0

1

2

4Bacteria in water samples

x 10

10 ce

lls /

l

Exp. 1 Exp. 240 8 12 0 12 h 0

10

20

30

raceway H2O x

1010

cells

/ l

• 70-80% of all bacteria consisted of members of Bacteroidetes,α- and γ-proteobacteria

• Vibrio spp. dominate feces flora, but Bacteroidetes and α-proteobacteriagrew about twice as fast (5.3 h vs 3.0 h and 2.6 h, respectively)

• Different microbial community structure in water than in feces

Bacterial Diversity

0.0

17.5

35.0

52.5

70.0

FP 0h FP 12h H2O 12h

Bacteroidetes (CF319a) α-proteobacteria (ALF968) γ-proteobacteria (GAM42a) Vibrio spp. (GV822)

% o

f tot

al c

ells

µmol

/ (g

*h)

0

8

15

23

30

h0 12

phosphatase aminopeptidase ß-glucosidase

Microbial Hydrolytic Ectoenzyme Activities

•Phosphatase and aminopeptidase activities extremely high ! 102x higher than in mesotrophic coastal waters

• Aminopeptidase: use protein as N source • Alkaline Phosphatase: use organo-phosphorous compounds for engery; recycle P

Phosphatase, Aminopeptidase, and β-Glucosidase Activities in Feces

0

2

4

6

8

Phosphatase, Aminopeptidase, and β-Glucosidase Activities in Water Column

µmol

/ (l*

h)

0.0

1.8

3.5

5.3

7.0

h0 4 8 12

phosphatase L-aminopeptidase ß-glucosidase

•Phosphatase and aminopeptidase activities extremely high

àindicates availability of substrates in water column

raceway

Microbial Hydrolytic Ectoenzyme Activities

µmol

/ (g

*h)

0

3

6

9

12

0 12

a-glucosidase chitobiaselipase

h

µmol

/ (g

*h)

0

1

1

2

2

0 12

a-glucosidase chitobiase lipaseh

Feces Water samples

• Extremely high chitobiase activity indicates utilization of peritrophic membrane

! transfer of chitin-N into bacterial protein ! utilizable for shrimp again

• α-Glucosidase and lipase activities also 102 higher than coastal waters ! if feed stays too long in water, valuable omega-3 fatty acids might be lost

Microbial Hydrolytic Ectoenzyme Activities

α-Glucosidase, Chitobiase, and Lipase Activities

Summary of Results

1. Shrimp feces are “hot spots” of bacterial production exhibiting explosive growth rates.

2. Fecal bacteria “seed” the water column with bacteria where concentrations can exceed 1011 cells/mL.

3. Microbial community structure is different between water column and feces.

4. Phosphatase and aminopeptidase activities extremely high in feces and water, whereas glucosidase activity is low.

5. Bacteria enrich protein content of feces to 80% of feed within 24 hours. Coprophagous shrimp can recycle lost N.

Bacteria

Feces

DOMPOM

Feed

(flocs,fragments) (DOC,DON)

(20g)

(100%)

17.6%86.3%

0.8% 3.6%

1.1%

12.9%

Mass Balance of Shrimp Feed

~ 14% of shrimp feed is lost as DOM or POM.

Can shrimp recycle N lost through DOM via microbial loop?

Novel Feeding Mechanism

➢Observed “sweeping” behavior of 3rd maxillipeds with the two diatoms.

Scanning EM photos of maxillipeds

➢3rd maxillipeds may form a filter-feeding “net”’ with a mesh size of ~10 µm in 2-g shrimp.

➢ This “net” may trap floc-associated bacteria, thereby recycling “lost” N.

Kent et al. Aquaculture, 319 (2011) 363–368

Research Program

Short term

1.Using molecular-based approaches, characterize microbial community structure in biosecure, intensive production systems and quantify spatial and temporal variability of these communities.

2.Identify candidate "effective microorganisms" (EM) to use in a defined inoculum to increase system productivity and profitability.

Culture-independent technologies and the application of –omics will drive a new view of microbial diversity and function in aquaculture systems and allow for a process-oriented ecosystem approach to studying microbial community structure and function.

Research Program

Mid-term

1.Using biosensor technology and fluorescence sensing instrumentation, identify critical control variables that impact microbial community structure and function. 2.Develop best management strategies to maintain the persistence of beneficial microbes contained in the EM inoculum. 3.Operate a successful, large-scale biosecure, intensive shrimp production system (with a defined microbial inoculum, specialty feeds, and genetically improved shrimp) that can produce large amounts of shrimp routinely and predictably with low risk of system failure.

Research Program

Long term

1.Using transgenic technology, create a defined microbial inoculum with desirable characteristics that can out-perform an EM inoculum derived from in situ microorganisms.

2.Develop mathematical models which integrate interactions of inoculum microbes with exogenous system inputs so that system outputs can be predicted and maximized.

Slide 3

P h y t o p l a n k t o nP r o t o z o aB a c t e r i a

A l k a l i n i t y

F e e d

DOM(DOC, DON)

Shrimp

F r a g m e n t s

FecesF l o c

CO2NH4+NO2-

N2O

N2

NO3-

Viruses

Unmasking the Microbial Black Box Integrative view of microbial processes

in shrimp production systems

Technical Information Flow

Basic Science → Applied Science → Extension Agent → Shrimp Producer

→

There is an urgent need to establish an objective, science-based extension service for the shrimp farming industry, supported by national governments or regional bodies, which translates basic and applied scientific knowledge to the farmer in an effective and meaningful way.

Thank You!