IMP - MSC - Nils - Sommer - Artificial Artemia

Evaluation of Nematodes and Artificial
Artemia as Feed for Pacific White

Shrimp in a Biofloc Nursery System
Nils Phillip Sommer
A Master Thesis in Aquaculture and Fisheries
Faro, January 2019

Artemia as Feed for Pacific White Shrimp in
a Biofloc Nursery System
Nils Phillip Sommer
Dissertation for the Attainment of the Master’s Degree in

Aquaculture and Fisheries
Thesis was supervised by Dr. Cláudia Aragão

Centro de Ciências do Mar (CCMAR)
Universidade do Algarve, Faro, Portugal
and
Dr. Felipe do Nascimento Vieira
Universidade Federal de Santa Catarina, Florianopolis, Brazil
Faro, January 2019
I
Artemia as Feed for Pacific White Shrimp in
a Biofloc Nursery System
Declaração de autoria de trabalho
Declaro ser o autor deste trabalho, que é original e inédito. Autores e

trabalhos consultados estão devidamente citados no texto e constam na
listagem de referências incluída.
Universidade do Algarve, 30 Janeiro de 2019
________________________________________
© 2019 Nils Phillip Sommer
A Universidade do Algarve tem o direito, perpétuo e sem limites geográficos, de arquivar e

publicitar este trabalho, através de exemplares impressos reproduzidos em papel ou de
forma digital, ou por outro meio conhecido ou que venha a ser inventado, de o divulgar
através de repositórios científicos e de admitir a sua cópia e distribuição como objetos
educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e
editor.
II
Acknowledgments
Foremost, I would like to thank Burkhard Hormann and the Colossal Fish GmbH for
establishing the initial contacts to the Laboratório de Camarões Marinhos that lifted this
project into existence. Their financial support to cover the expenses and materials is
highly appreciated. Furthermore, I like to thank my supervisor Dr. Felipe Vieira of the
Universidade Federal de Santa Catarina for accepting this project at his laboratory and
for his help with the experimental design and the management of the logistical and
practical challenges that arose. A thank you is also due to my supervisor Dr. Cláudia
Aragão from the Universidade de Algarve, who helped me with the theoretical part of
the thesis and provided invaluable and precise corrections to the manuscript. Special
gratitude goes to Dr. Laurent Seychelles and the E-nema GmbH, who provided treasured
information about nematodes and who went through unforeseen troubles in supplying
me with a much-needed sample of their protégés.
Very special thanks go to Priscila Costa Rezende, who supported me in the experimental
setup, the daily activities around the laboratory, and who is the main reason why I
practically speak Portuguese now. I would also like to thank Néa, Patri, and Vitor for
helping with the almost around-the-clock feeding and sampling tasks that this project
brought upon us. Moreover, I would like to express my sincere appreciation to all the
unnamed employees and students at the LCM who helped me with this experiment
through myriad tasks. This project would not have been possible, much less successful,
without the assistance of many active hands.
My deepest gratitude also goes to my parents for their unfailing support throughout my
studies, who spared no effort to witness the final days of this experiment in the most
beautiful part of Brazil. Last but not least, I would like to thank Janna Hilz for
proofreading and correcting the language of this document and for making the overall
experience on the Island of Santa Catarina unforgettable.
III
Abstract
The global aquaculture production is growing immensely in all aspects and has already
surpassed the output from wild caught fish and shellfish industries. The farming of
Penaeus vannamei is one of the biggest contributors to this market. But many early
stages of aquaculture depend on the finite and volatile resource Artemia as a live feed.
This dependency has been identified as a bottleneck for future growth and sustainability
progress. In this experiment, one artificial Artemia product and one nematode species
were tested and evaluated in a feeding trial as potential replacements for live Artemia in
a zero-water exchange biofloc nursery system. P. vannamei post larvae (PL) were stocked
at a density of 60 PL/L in 60-L tanks. They were reared from PL12 – PL33 and fed 8 times
per day with a dry feed (DF). Different treatments with four replicates each received a
diet supplement of either live nematodes (N), live Artemia (LA), artificial Artemia (AA),
or DF (control, C). The PL that received the live diets had nearly twice the survival rate
compared to the ones only fed inert diets (N: 94 ± 6%, LA: 91 ± 7%, AA: 53 ± 11%, C: 51 ±
10%). Growth parameters were slightly better in the two inert diet groups (wet weight:
N: 22.5 ± 5 mg, LA: 22.5 ± 5 mg, AA: 35 ± 5.7, C: 35 ± 10; Total length: N: 15.8 ± 3.9 mm,
LA: 15.8 ± 3.2, AA: 17.1 ± 5.1 mm, C: 17.3 ± 5.0). No significant differences were detected
in survival to salinity stress. In addition, beneficial effects on the biofloc and on the water
quality were observed in the live diet groups and their causation should be further
investigated. The results show that the nematode species Panagrolaimus sp. (NFS 24-5)
can completely replace live Artemia in a co-feeding regime.
Keywords: Penaeus vannamei, nematodes, artificial Artemia, biofloc, nursery
IV
Resumo
Nas últimas décadas a produção aquícola global experimentou um imenso crescimento

em quantidade, variedade de espécies cultivadas e sistemas de produção. Desde 2014 que
a aquicultura ultrapassa a produção mundial de peixes e mariscos capturados. O
crescimento sem precedentes da população humana é o principal motor de uma procura
crescente de proteína aquática de alta qualidade. O cultivo do crustáceo marinho
Penaeus vannamei é um dos maiores contribuintes para este mercado. No entanto as
fases iniciais das produções de muitos moluscos e peixes dependem de Artemia como
alimento vivo, um recurso finito e volátil. Os cistos de Artemia só podem ser colhidos
em algumas regiões do planeta e sua produção está sujeita a grandes flutuações naturais,
o que limita sua exploração contínua. A dependência de Artemia por parte da indústria
da aquicultura foi identificada como um estrangulamento ao crescimento e progresso
do sector. A grande procura de proteína aquática levou a uma enorme pressão sobre
muitos recursos naturais, em todos os tipos de ecossistemas aquáticos, desde os oceanos
às águas interiores e passando pelos ambientes costeiros. A resposta da aquicultura tem
de ser persuasiva no sentido de dar garantias de sustentabilidade, mantendo as suas
atividades dentro de limites saudáveis. A obtenção de autonomia relativamente à
Artemia poderá simplificar significativamente os processos de alimentação durante as
fases larvares e, eventualmente, promover a sustentabilidade da aquicultura em geral.
Os produtos artificiais de Artemia já estão comercialmente disponíveis mas sua

capacidade de alimentar eficientemente larvas de camarão ainda carece de testes
científicos. Mais recentemente, uma espécie de nematode foi identificada como tendo
propriedades semelhantes às dos cistos de Artemia: armazenamento a longo prazo em
estado desidratado, processo de reidratação simples, tamanho pequeno para fácil
ingestão e valor nutricional adequado para larvas de camarão. Além disso, estão
disponíveis métodos de produção em massa, sustentáveis e economicamente viáveis,
que facilitam sua aplicação comercial.
Nesta experiência, um produto artificial de Artemia e uma espécie de nematode foram
testados e avaliados num ensaio de alimentação como substitutos potenciais de Artemia
V
viva num sistema de viveiro biofloco sem troca de água. Os sistemas Biofloc dependem
do controle de nitrogénio através de processos microbianos que ocorrem dentro do
tanque de cultivo, o que reduz substancialmente a renovação de água com o benefício
adicional de fornecer proteína bacteriana. As pós-larvas (PL) de P. vannamei foram
estabuladas com uma densidade de 60 PL por L em tanques semi-cónicos de 60 L. As
larvas foram criadas a partir de PL12 - PL30 e alimentadas 8 vezes por dia com ração seca.
Diferentes tratamentos com quatro replicados receberam um suplemento alimentar de
nemátodos vivos (N), Artemia viva (LA), Artemia artificial (AA) ou ração seca (controle,
C). As PL que receberam as dietas vivas tiveram quase o dobro da taxa de sobrevivência
quando comparadas às alimentadas apenas com dietas inertes (N: 94 ± 6%, LA: 91 ± 7%,
AA: 53 ± 11%, C: 51 ± 10%). No entanto, os parâmetros de crescimento foram ligeiramente
melhores nos dois grupos de dieta inerte (peso húmido: N: 22,5 ± 5 mg, LA: 22,5 ± 5 mg,
AA: 35 ± 5,7, C: 35 ± 10; Comprimento total: N: 15,8 ± 3,9 mm, LA: 15,8 ± 3,2, AA: 17,1 ± 5,1
mm, C: 17,3 ± 5,0). O efeito do stress salino não foi significativo. Nos grupos de dieta viva
foram observados efeitos benéficos na formulação de bioflocos com reduzida
acumulação de sólidos suspensos, totais e voláteis.
A dieta de nematode também teve níveis de nitrito significativamente menores,

indicando melhor controlo do nitrogénio e parâmetros gerais de qualidade da água. O
contexto e a causalidade dessas diferenças devem ser investigados, pois podem ser
relevantes para operações de aquicultura usando sistemas de bioflocos. Em conclusão, a
experiência verificou que a espécie nematode Panagrolaimus sp. (NFS 24-5) pode
substituir completamente Artemia viva num regime de co-alimentação. Os resultados
deste estudo de alimentação podem promover a sustentabilidade da criação de camarão
por meio de alimentos alternativos, independentes dos recursos finitos de Artemia.
Palavras-chave: Penaeus vannamei, nematodes, Artemia artificial, biofloco, viveiro
VI
List of Abbreviations
AA Artificial Artemia m Meter

[AA] Artificial Artemia Group mg Milligram
AOB Ammonia Oxidizing mm Millimeter
Bacteria NH3 Ammonia, unionized type
ANOVA Analysis of Variance n Numbers, Sample Size
APHA American Public Health [N] Nematode Group
Association N Nitrogen
ARA Arachidonic Acid NH4+ Ammonia, ionized type
BFT Biofloc Technology NO2- Nitrite
°C Degree Celsius NO3- Nitrate
C Carbon NOB Nitrite Oxidizing Bacteria
[C] Control Group NV Naamloze Vennootschap,
CO2 Carbon dioxide Belgium Limited Company
DHA docosahexaenoic Acid O Oxygen
DO Dissolved Oxygen p p-value, Probability Value
DF Dry Feed PL Post Larvae
EPA Eicosapentaenoic Acid PVC Polyvinyl-Chloride
EU Experimental Unit RAS Recirculating Aquaculture
FAO Food and Agriculture System
Organization RGR Relative Growth Rate
g Gram SPF Specific Pathogen Free
h Hour SPR Specific Pathogen Resistant
H Hydrogen t Time
HSD Honest Significant TAN Total Ammonia Nitrogen
Differences TSS Total Suspended Solids
IPCC Intergovernmental Panel UFSC Universidade Federal de
on Climate Change Santa Catarina
L Liter VSS Volatile Suspended Solids
[LA] Live Artemia group W0 Initial Wet Weight
LCM Laboratório de Camarões Wt Final Wet Weight
Marinhos WSSV White Spot Syndrome Virus
LTDA Limited Company YSI Yellow Springs Instruments
VII
Table of Contents
1. Introduction .......................................................................................................................................... 1
1.1. Aquaculture Overview.................................................................................................................. 1
1.2. Penaeus vannamei ....................................................................................................................... 2
1.3. History of Penaeus vannamei Aquaculture ................................................................................... 3
1.4. Production Techniques of Penaeus vannamei............................................................................... 4
1.5. Current Issues and Measures........................................................................................................ 7
1.6. Biofloc Technology ....................................................................................................................... 9
1.7. Artemia ...................................................................................................................................... 11
1.8. Nematodes ................................................................................................................................ 13
1.9. Objectives and Goals .................................................................................................................. 15
2. Material and Methods........................................................................................................................... 1

2.1. Preparation of the Experimental Facility..................................................................................... 16
2.2. Feeding Trial............................................................................................................................... 19
2.3. Feed Preparation........................................................................................................................ 21
2.3.1. Preparation of live Artemia ........................................................................................................... 21
2.3.2. Preparation of Nematodes ....................................................................................................... 23
2.3.3. Preparation of dry feed and artificial Artemia .............................................................................. 23
2.4. Water Quality Analysis ............................................................................................................... 24
2.5. Salinity Stress Test ..................................................................................................................... 25
2.6. Final Sampling ............................................................................................................................ 25
2.7. Statistical Analysis ...................................................................................................................... 26
3. Results ................................................................................................................................................ 27
3.1. Water Quality Parameters .......................................................................................................... 27
3.1.1. Temperature and Salinity .............................................................................................................. 27
3.1.2. Dissolved Oxygen .......................................................................................................................... 28
3.1.3. Alkalinity ....................................................................................................................................... 29
3.1.4. pH .................................................................................................................................................. 30
3.1.5. Total Suspended Solids ................................................................................................................. 32
3.1.6. Volatile Suspended Solids ............................................................................................................. 33
3.1.7. Total Ammonia Nitrogen............................................................................................................... 34
3.1.8. Nitrite ............................................................................................................................................ 35
3.2. Growth Performance .................................................................................................................. 36
3.2.1. Survival .......................................................................................................................................... 36
3.2.2. Growth ..................................................................................................................................... 37
3.2.3. Body Composition ......................................................................................................................... 38
VIII
3.2.4. Salinity Stress Test .................................................................................................................... 39
4. Discussion ........................................................................................................................................... 40
4.1. Water Quality Parameters .......................................................................................................... 40
4.2. Larval Performance .................................................................................................................... 46
4.3. Final Conclusions ........................................................................................................................ 50
5. References .......................................................................................................................................... 51
Table of Figures
Figure 1: Experimental Units and Overview of the Wet Laboratory Room ..................... 17
Figure 2: Holding Tank ........................................................................................................ 18
Figure 3: Artemia Hatching Tank. ...................................................................................... 22
Figure 4: Temperature......................................................................................................... 27
Figure 5: Salinity .................................................................................................................. 28
Figure 6: Dissolved Oxygen ................................................................................................ 29
Figure 7: Alkalinity .............................................................................................................. 30
Figure 8: pH .......................................................................................................................... 31
Figure 9: Pearson Correlation of Alkalinity and pH ......................................................... 31
Figure 10: Total Suspended Solids ...................................................................................... 32
Figure 11: Volatile Suspended Solids................................................................................... 33
Figure 12: Total Ammonia Nitrogen ................................................................................... 34
Figure 13: Nitrite .................................................................................................................. 35
Figure 14: Final Survival of P.vannamei Post Larvae ......................................................... 36
Figure 15: Post larvae Body Composition ........................................................................... 38
Figure 16: Stress Test Survival of P.vannamei Post Larvae ............................................... 39
Table of Tables
Table 1: Feeding Schedule. .................................................................................................. 20

Table 2: Amount of Added Diet, Dextrose, and Calcium Hydroxide ............................... 21
Table 3: Growth Results of P. vannamei Post Larvae. ....................................................... 37
IX
1. Introduction
1.1. Aquaculture Overview
In the last decades, the global aquaculture production experienced immense growth in
total quantity, the variety of farmed species, and applied production systems (Bostock
et al., 2010; Engle et al., 2017). In the year 2014, the total volume of farmed aquatic
animals matched the total of wild caught fish and shellfish for the first time in history
(FAO, 2016). This vast growth in output is largely driven by the urgent and imminent
need to feed an ever-increasing human population of already 7.5 billion people to date,
estimated to reach close to 9.5 billion in the year 2050 (United Nations, 2017). Along
with this unprecedented growth in the total population emerges a soaring demand for
high-quality protein, especially from aquatic sources. On a global average, mankind's
growing appetite for fish and shellfish has already reached an annual consumption of
more than 20 kg per capita (FAO, 2016). Estimates from the World Bank, the United
Nations, and the Food and Agriculture Organization concluded that by 2030 over 60
percent of the consumed fish and shellfish will be provided by aquaculture (The World
Bank, 2013). This shift towards more contribution from aquaculture to human nutrition
will most likely continue hence after. Effects such as ocean warming and acidification,
attributed to ongoing anthropogenic greenhouse gas emissions as most recently and
prominently presented by the Intergovernmental Panel on Climate Change (IPCC, 2018),
may lead to drastic redistributions of wild catch potentials in the global oceans with
possible negative implications for many regions (Cheung et al., 2010). Particularly
affected will most likely be the lower latitudes and the tropics, where also the fastest
population increase and, hence, the highest requirement of fish as food is expected.
The high demand for aquatic protein, which has led to enormous pressure on many
natural resources, from offshore oceans - through coastal environments - to inland
freshwater systems, must be met with a persuasive movement towards more
sustainability and nature conservation to keep exploitation of wild catches and
1
aquaculture activities in prospering and healthy limits. Therefore, in the face of global
population growth and climate change, the world is required to find ways to produce
enough food to feed mankind while preserving the planets natural habitats. In this
scenario, especially the relatively new industry of aquaculture has been identified as a
food producing sector with immense potential in growth and innovation. If aquaculture
further continues towards more sustainability, it will play a significant role in the
provision of nutritional and economic needs for future generations (Diana, 2009).
One important production segment of aquaculture is the group of marine crustaceans,

with a contribution of almost 10% to the global market. And within this group, the
species Penaeus vannamei (P. vannamei, Boone), commonly known as the Pacific white
shrimp or whiteleg shrimp, accounts for over 80% of all farmed marine shrimp (Dugassa
& Gaetan, 2018; FAO, 2016; Zhang et al., 2019). When considering all produced species
in the world aquaculture industry, P. vannamei was ranked 6th in 2016 with a total
volume of over 4 million tons (FAO, 2018).
1.2. Penaeus vannamei
In the late 1990s, a controversial taxonomic revision proposed by Pérez Farfante &
Kensley (1997) split the former monophyletic Penaeus group into six genera, one of
which was the genus of Litopenaeus, containing the species vannamei. Though, more
recent genetic and molecular analysis of the Penaeid shrimp refutes the six-genus
classification, resulting in a now widely accepted resumption of the former genus and
species name: Penaeus vannamei (Ma et al., 2011).
Originally, P. vannamei is native to the tropical marine habitats of the Eastern Pacific,
ranging from northern Mexico through Central America until North Peru. Water
temperatures in this region are usually above 20-25 °C all year round (Dugassa & Gaetan,
2018). Adults live near or offshore in depths reaching up to 70 m and mate and spawn in
the open ocean. The life-cycle of P. vannamei is rather complex and passes through
2
several morphologically differing stages. Maturity is attained with approximately 6-7
months of age when males have reached over 20 g and females over 28 g of body weight.
A full-grown female can spawn up to a quarter million eggs at once, which are fertilized
by male sperm in the external environment. About 16 hours after spawning and
successful fertilization, the first larval stages, called nauplii, hatch from the eggs. The
nauplii can swim intermittently and are independent of food but rely on internal yolk
sack reserves as nutrition. After a few days, through metamorphic processes, the first
larval stage is developed into the protozoa, which actively feeds on phytoplankton and
unicellular algae. Further development phases lead through the mysis and early
postlarvae (PL) stage, both of which mainly prey on zooplankton, such as rotifers,
Artemia, and copepods. The late PL stage is already very similar in morphology to
juvenile and adult stages. An adult will continue to grow and experience on average 50
molting periods during its lifetime (Dugassa & Gaetan, 2018; Zhang et al., 2019). After
the final molting into a PL, the planktonic habitat is left behind, and migration inshore
occurs passively through ocean currents and actively through impulsive swimming
motions. In coastal waters, the PL find shelter and prey in mangroves, lagoons, and
estuaries. During their benthonic adult life, the omnivorous P. vannamei feeds on
detritus, worms, bivalves, and other crustaceans (Bailey-Brock & Moss, 1992). There is
also evidence of sporadic cannibalistic behavior when they occur in high densities,
which is usually only the case in captivity or aquaculture production (Romano & Zeng,
2017). This behavior has also been reported for several other penaeid shrimp species,
such as P. monodon and P. esculentus (Abdussamad & Thampy, 1994; Arnold et al., 2005).
1.3. History of Penaeus vannamei Aquaculture
The relatively young aquaculture history of P. vannamei started in the 1970s when
specimen caught from the wild off the coast of Panama were successfully spawned by
French scientists in Tahiti and the species' life-cycle was closed for the first time (Michel
et al., 2013). Further research achieved advances in the predictable maturation
promotion through unilateral eyestalk ablation of females, which induces a hormonal
3
cascade affecting all aspects of the shrimp’s physiology and eventually leads to spawning
(Kannan et al., 2015). Also, the enhanced and specialized nutrition of the broodstock has
improved the spawning of eggs in quantity and quality (Wouters et al., 2001). Intensive
breeding techniques and genetic domestication programs eventually led to widespread
shrimp aquaculture applications in the Hawaiian Islands, South United States of
America, and many Central and South American countries. From the 1980s the
commercial production in Latin America was growing rapidly but was periodically
diminished by disease outbreaks, usually during the colder La Niña years. In the late
1980s, specific pathogen free (SPF) strains were developed at the Oceanic Institute in
Hawaii, that reduced the impact of certain diseases and supported further shrimp
farming activities (Wyban & Sweeney, 1991). In the late 1990s, P. vannamei was also
introduced in the Asian aquaculture industry as an alternative to the major indigenous
species P. monodon and P. chinensis, with which many farmers had disease susceptibility
issues (Briggs et al., 2004). Since the implementation of P. vannamei in Asia, the species
has experienced an immense increase of production until today, with China now being
by far the biggest player worldwide and current producer of over 1 million tons annually
(FAO, 2016).
1.4. Production Techniques of Penaeus vannamei
The aquaculture production of P. vannamei is fundamentally based on the grow-out of

larvae obtained from a broodstock, which was originally caught in the wild or, as
nowadays is the norm, has been domesticated and genetically improved over many
generations in a specialized facility. Domestication has resulted in the production of
broodstock shrimp strains with desirable traits, such as faster growth, lower salinity or
temperature tolerance, or disease resistance. Significant success and a step forward was
the development of specific pathogen free (SPF) and specific pathogen resistant (SPR)
shrimp lines, which helped to overcome early issues with certain disease outbreaks
(Browdy, 1998). The animals used as broodstock are reared, either individually or in
groups, in so-called maturation tanks with optimal husbandry conditions comprised of
4
filtered seawater, fresh and specialized broodstock feeds, optimal photoperiod and
temperature, and under continuous care by trained personnel. One eyestalk of the
females is ablated, which will induce a repeated spawning event through a hormonal
cascade. After successful spawning and fertilization by the males, the freshly hatched
and positively phototactic nauplii are concentrated with a light source and transferred
to a larval rearing tank or hatchery tank. In the ensuing hatchery phase, the nauplii are
usually reared in "U" or "V"-shaped tanks with pure oxygen or air supply introduced
from the bottom in order to keep the larvae afloat and emulate their planktonic life
stage. They are predominantly fed with live feed, microalgae, and Artemia, often
supplemented by liquid or dry formulated diets. The water is regularly exchanged at a
rate of more than 100% per day to ensure optimal water conditions. When the larvae
reach the PL10-12 stage, they are transferred directly to the grow-out tanks or an
intermediate nursery tank system. In the shrimp aquaculture industry, the PL10 stage,
for e.g., is defined as an animal that went through all phases of larval development after
hatching; from nauplii (1-2 days), zoea (3-5 days), mysis (3-5 days), until it reaches the
PL stage, plus the 10 days it has already lived as a PL. This means that a PL10 is actually
between 17 and 22 days old, depending on the development time in each pre-PL stage
(Dugassa & Gaetan, 2018).
When a nursery system is applied before the grow-out phase, the PL are reared for one
to five weeks with relatively high density in tanks that can be easily monitored and
controlled for their water conditions. This ensures higher survival and faster growth in
the early PL phases and leads to stronger PL that can then be transferred to the harsher
conditions of grow-out ponds. This technique is especially applied in colder areas with
shorter growing seasons, where the nursery tanks are set up in greenhouses or have
heated water (Briggs, 2006).
The final grow-out systems can be classified as extensive, semi-intensive, intensive, and
super-intensive, mainly depending on the densities of stocked PL. The simplest
technique, largely applied in developing countries, is the extensive culture in irregularly
shaped, earthen ponds that can be flooded and drained with the tidal flow. None or only
minimal pumping and aeration is applied, the stocked PL are mainly fed on naturally
5
occurring prey within the pond, and only small and few shrimps can be harvested at the
end of a grow-out season. In semi-intensive systems, larger densities are stocked, the
water is exchanged regularly by pumping, the shrimp are fed several times daily with
formulated diets, and the final yield, but also the investment and workload is
considerably higher (Briggs, 2006).
More technically sophisticated intensive farms have ponds covered with a plastic lining,
concrete raceways or circular tanks in various dimensions that can be completely
drained, independent of the tides. Often times, these farms are located away from the
coast on budget-priced land and use non-oceanic water sources with lower salinities.
The densely stocked ponds need to have heavy aeration to achieve sufficient
oxygenation and water movement. The shrimp are more intensely fed, and the water
must be renewed frequently and channeled to settling ponds in order to cope with
deteriorating water conditions. High renewal rates of water can be expensive,
deteriorating for the surrounding environment, and may be a source of viral diseases.
Alternatively, reduced water exchange systems, such as the biofloc technology (BFT),
are now widely applied to confront these issues. Nevertheless, rigorous biosecurity
measurements with careful monitoring and management of water, aeration, feed, and
stocked PL are inevitable to achieve high yields in intensive farming systems.
In world regions without favorable all year-around temperatures of above 29 °C, the
grow-out systems are located in greenhouses or even structured indoor facilities to help
maintain high water temperatures during colder periods. Due to the higher cost of
roofed space in more urbanized areas, PL are stocked in very high density in raceways
or circular tanks, so-called super-intensive rearing systems. Often times, these
enterprises rely on recirculating aquaculture systems (RAS) with a zero-water exchange,
where only the evaporated water is replaced, and all accumulated biological waste is
processed with biological filters. These systems are therefore considered to have high
biosecurity and a low ecological footprint while allowing to produce shrimp of good
quality in a sustainable manner. Recent technical advances in RAS have led to an
increased interest in their application at locations completely independent of oceanic
6
water sources, temperate climate and with close proximity to consumers interested in a
fresh and sustainably produced seafood.
1.5. Current Issues and Measures
One of the main issues that have arisen with the intensified large-scale shrimp
aquaculture is disease outbreaks that threaten to reduce yields to a minimum or wipe
out entire farms in affected regions. Vibriosis, a bacterial disease transmitted by various
species of Vibrio spp., is one of the major problems and responsible for high mortalities
in affected shrimp farms worldwide (Chandrakala & Priya, 2017). Vibrio spp. are naturally
occurring opportunistic bacteria that can become pathogenic when the shrimp’s
immune system is suppressed, which can be the case at high densities with poor water
and husbandry conditions. Many producers, especially in developing countries, have
been counteracting the outspread of diseases with the broad administration of
antibiotics during the production cycle (Bermudez-Almada & Espinoza-Plascencia,
2012). Indiscriminate usage of antibiotics in shrimp farms is associated with
environmental issues, such as bacterial resistance and persistence of the disease and
toxic residues in the water, sediment, and adjacent aquatic ecosystems. Farmers that are
handling antibiotics regularly and local communities nearby that are extensively
exposed to antibiotics in their environment and are in risk of suffering severe health
issues. Also, antibiotic residues in the edible parts of the shrimp may also negatively
affect the health of the consumers and have already led to concerns about food safety
(Bermudez-Almada & Espinoza-Plascencia, 2012; Grigorakis, 2010; Holmström et al.,
2003).
Another cause of widespread diseases in the shrimp aquaculture stems from the
occurrence of several different types of pathogenic viruses. One extremely virulent
species, among the 20 identified viruses affecting shrimp thus far, is the White Spot
Syndrome Virus (WSSV). At affected shrimp farms, it can cause very rapidly emerging
mortalities of up to 100% within 10 days. The financial losses in the 1990s alone, caused
7
by WSSV outbreaks worldwide, have been estimated to be worth more than 5 billion US
dollars (Ganjoor, 2015). However, viruses are a common and naturally occurring
biological agent in the marine environment, thus, their complete exclusion from
aquaculture systems is practically impossible. The transmission may occur vertically
from parent to the next generation, or horizontally from individual to individual.
Applied methods to combat infestation and dispersion include strict biosecurity
measures to avoid transmission, filtration and treatment of water sources, the usage of
PL from certified SPF strains, the augmentation of the shrimp’s natural immune
responses through probiotics and specialized feed, and good overall husbandry
conditions to reduce animal stress (Ganjoor, 2015; Walker & Mohan, 2009). A drastic
reduction of water exchange, as is possible with BFT and RAS, indoor facilities, and strict
feed controls can significantly reduce the risk of exposure to these disease agents, which
may otherwise use transmission vectors such as water, feed, intruding animals, and lack
of hygiene (Emerenciano et al., 2013).
A serious problem concerning the natural environment and the integrity of coastal
regions in the tropics is the vast destruction of invaluable and endangered mangrove
habitats through large constructions of shrimp farming ponds (Ashton, 2008; de Graaf
& Xuan, 1998; FAO, 2016). Further, many farms, especially in developing countries with
lax environmental protection laws and poor water management systems, have been
reported to release large volumes of effluents to their surroundings that contaminate
fresh and marine water habitats (Ahmad et al., 2017). The cumulative issues of diseases,
mangrove destruction, and environmental pollution in the shrimp aquaculture industry
preceded a decrease of consumer trust, especially in the sustainability and healthy food
conscious group (Grigorakis, 2010; Shepherd & Little, 2015). Therefore, modern,
biosecure, zero-water exchange systems located near urban areas may be able to reduce
the pressure on the natural environment by producing healthy shrimp without
antibiotics in the vicinity of the consumer. The absence of long transportation routes
would also reduce carbon footprint and allow the marketing of a fresh and unfrozen
high-value sustainable seafood product.
8
1.6. Biofloc Technology
The intensification of aquaculture brings with it an immense excess of organic

pollutants, feed residues and biological wastes that accumulate in the rearing water. In
addition to feces, aquatic organisms also excrete ammonia from their gills as the
nitrogen end product of the protein catabolism. Depending on the temperature,
alkalinity, and pH of the water, ammonia nitrogen can be present as the ionized type
(NH4+) or the unionized type (NH3). The sum of both types is usually referred to as the
total ammonia nitrogen (TAN) and is used as a limiting water quality parameter (Chen
et al., 2006). Ammonia is a small molecule, very soluble in water and permeates cell
membranes relatively easily (Wright, 1995). In intensive culture systems with a high
density of organisms and large biomass, the accumulation of ammonia in the water can
quickly reach toxic levels. The most common method to attend this issue is the
continuous replacement of the rearing water. This procedure requires very large
volumes of clean incoming water and also produces equally large volumes of polluted
effluents that deteriorate the adjacent environment. The RAS technology addresses this
issue, and, with the use of biological filters hosting autotrophic nitrifying bacteria that
oxidize ammonia to the lesser toxic nitrogen forms of nitrite and nitrate, only about 10%
of the total water volume needs to be replaced (Ahmad et al., 2017). Though, the large
financial investment and the high operational and maintenance costs of technology in
RAS is inhibiting the broad application of this approach, especially in developing
countries.
On the contrary, BFT has gained a lot of attention in recent years for its ability to control
toxic nitrogen accumulation in a cost-effective, simple, and sustainable way. Its
underlying principle of ammonia control is the promotion and maintenance of high
levels of heterotrophic bacteria through the manipulation of the carbon:nitrogen (C:N)
ratio in the water. By increasing the available carbon in the system, either by directly
adding carbon-rich carbohydrates or the use of low protein feed with a high C:N ratio,
heterotrophic bacterial growth is stimulated and nitrogen is assimilated through
microbial protein metabolism (Ahmad et al., 2017; Avnimelech, 1999). The fast-growing
9
heterotrophic bacteria rapidly develop into dense communities of microorganisms and
algae, which establish a flocculated suspension in the water, also called biofloc. This
biofloc functions as a bioreactor controlling the water quality and can also serve as an
additional source of protein for grazing shrimp (Avnimelech et al., 1994). Within a
mature biofloc, toxic TAN and nitrite (NO2-) can be immobilized by heterotrophic
assimilation into bacterial biomass and by autotrophic nitrification from TAN to nitrite
and, in a further step, to nitrate (NO3-). When the C:N ratio is elevated to 15-20:1, the
excreted nitrogen becomes the limiting growth factor and the heterotrophic bacteria
readily incorporate it into their cells and thereby remove it from the water (Avnimelech,
1999). Compared to autotrophic nitrifying bacteria, which are primarily responsible for
ammonia breakdown in RAS biofilters, heterotrophic bacteria have a 10 times higher
growth rate and will, therefore, become the predominant bacteria in an environment
with a high C:N ratio. Thus, toxic nitrogen species are immobilized much faster and
more efficiently in a system with primarily heterotrophic bacteria (Hargreaves, 2006).
The microbial processes, described by Avnimelech (1999) and Ebeling et al. (2006) in
great detail, are taking place inside the rearing water, and are therefore independent of
external biofilters and pumping systems, significantly reducing system space and cost.
Due to the extremely fast growth of heterotrophic bacteria in carbon-rich conditions,

large amounts of bacterial biomass are produced and the accumulation of total
suspended solids (TSS), which includes the volatile suspended solids (VSS) of organic
origin, must be monitored and managed accordingly. It has been observed that TSS
levels in the range of 400-600 mg/L are the most beneficial for shrimp rearing in BFT as
they create favorable factors for the system stability and nitrogen control while
providing a healthy environment for the shrimp. Extreme high TSS levels of >800 mg/L
can lead to occlusions of the gills and lower the overall performance and survival of
shrimp in such systems, while low levels of <200 mg/L are insufficient in controlling the
TAN accumulation (Schveitzer et al., 2013). Another adverse effect of the fast increase of
organic matter ignited through the addition of carbohydrates to the system is the
corresponding increase of carbon dioxide (CO2) in the water. High levels of CO2 need to
be avoided and must, therefore, be compensated through the elevation of dissolved
oxygen (DO), which is essential to support the aerobic microbiological processes and
10
husbandry conditions for the shrimp. Hence, constant and sufficient aeration of the
rearing water is extremely important in BFT, in order to maintain elevated DO levels
and a well-mixed biofloc solution (Hargreaves, 2006).
The heterotrophic bacterial reaction also consumes a modest amount of alkalinity as an

additional carbon source, therefore continuously reducing the alkalinity level in the
water. Water sources with initial low alkalinity may require the addition of external
carbon, such as sodium bicarbonate or calcium hydroxide. To support a sufficient and
constant heterotrophic bacterial assimilation of nitrogen, the alkalinity levels should be
kept between 100-150 mg/L (Ebeling et al., 2006).
To start and stimulate the initial biofloc formulation, a calculated amount of bottom soil
containing beneficial microorganisms, ammonium sulfate, and carbon sources have to
be added to the water. A preferable alternative, where possible, is the inoculation of a
new biofloc with 30-50% water from an already existing and well performing mature
biofloc solution (Ahmad et al., 2017; Schveitzer et al., 2017).
1.7. Artemia
The natural diet of shrimp larvae generally consists of diverse phytoplankton,

zooplankton and biofloc compositions with various sizes and different nutritional
values. But the feeding of P. vannamei larvae in commercial hatcheries, usually only
consists of a few microalgae species and Artemia spp. nauplii as the main source of
animal protein (de Lourdes Cobo et al., 2015). During the following production stages,
the feeding exclusively with dry feed is common practice. Though, the quantity and
quality of the nutritional diet offered to the shrimp larvae and juveniles have large
implications for the further growth and development in the ensuing life stages. It has
been reported that a supplement of live Artemia during the nursery phase is beneficial
and produces larger PL that are more resilient and perform better in the grow-out phase
(Zelaya et al., 2007).
11
The brine shrimp Artemia is an extremophile micro-crustacean that naturally occurs in
hypersaline environments and has become a very popular live food for many aquaculture
enterprises involved in the production of early stages of marine fish and shellfish
(Gajardo & Beardmore, 2012). Artemia have a good nutritional value, are small and can,
therefore, be readily ingested by the larvae Its dormant cysts can be stored for extended
periods. The short incubation time of 24 hours to convert the desiccated cysts into free-
swimming nauplii make them a convenient live food that requires relatively little labor
and equipment for preparation (Sorgeloos et al., 1998). Starting with the emersion of
hatchery operations in the 1970s, the overall positive attributes of Artemia have made it
a highly sought-after essential in the aquaculture industry. But regrettably, the global
supply of this natural resource is not sufficient to support the ongoing growth of this
industry (Lavens & Sorgeloos, 2000). Artemia cysts can only be harvested in very few
regions on the planet, mostly in hypersaline inland salt lakes or man-managed salt
extraction ponds. Currently, the largest fishery of Artemia franciscana, the most
abundant and most used species of brine shrimp, is located in the southern arm of the
Great Salt Lake in the USA. This region alone contributes over 50% to the global Artemia
cyst production, although precise data on production statistics are lacking (Calderon et
al., 2004; Litvinenko et al., 2015; Mechaly et al., 2013). This condition has led to a
dependency of the world aquaculture industry on a single location and exporting
country (Sorgeloos et al., 1998). Additionally, the cyst production in the Great Salt Lake
is subject to very dynamic natural fluctuations, limited by relatively strict environmental
regulations, and prone to be affected by climatic changes. Recent research has also
revealed that the long-term selective harvest of buoyant cysts may cause unforeseen
evolutionary changes of A. franciscana, which could further challenge the sustainable
management and future productivity of this fishery (Sura & Belovsky, 2016).
The accumulated inconsistencies have led to supply shortages and volatile prices of
Artemia cysts in the past, affecting most of all marine aquaculture hatcheries and was
therefore identified by researchers as one of the major bottlenecks for future growth
(Lavens & Sorgeloos, 2000; Sorgeloos et al., 2001). To cope with this unreliability, other
locations and different species of Artemia, such as the harvest of A. persimilis in Chile
and Argentina or the aquaculture of the non-endemic A. franciscana in artificially
12
constructed salt ponds in the Mekong Delta of Vietnam, are being explored and
investigated. Though, further research is needed to confirm their potential in
commercial-scale applications that could contribute significantly to the ongoing global
demand of Artemia cysts (Le, 2018; Mechaly et al., 2013).
To ensure the future sustainability of marine aquaculture production, the search for
alternatives or complete substitutes of Artemia as live diets for the early larval stages is
ongoing. Besides the adequate nutritional values, live diet alternatives must meet certain
standards, which include the correct particle size, similar buoyancy in the water column,
stability, minimal loss of nutrients through leaching, high digestibility, and the
capability to be stored for long periods (Zelaya et al., 2007). Several products are already
commercially available and some research has even pointed to potential nutritional and
economic benefits when used in co-feeding regimes with live Artemia. But their broad
application as a complete replacement for a live feed in all production phases has to be
further investigated and published in peer-reviewed literature (Calderon et al., 2004;
Gamboa-Delgado & Le Vay, 2009).
The main requirements by predatory shrimp and marine fish larvae to its prey are the
attractiveness, the nutritional value, and especially the size. The successful uptake of
diet particles is mechanically restricted by the size of the predators feeding apparatus,
and thus, the prey must be small enough to be ingested (FAO, 1996). Artemia greatly
fulfills this requirement, as well as certain roundworms of the phylum Nematoda that
have caught the interest of scientists for various advantages.
1.8. Nematodes
Nematodes are an extremely diverse animal group that is found in all imaginable
habitats including freshwater, marine, or terrestrial environments. Their ecological
niches range from plant or animal parasites and predators to free-living non-parasitic
forms that mainly feed on bacteria. Nematodes also occur naturally in biofloc and
13
Brüggemann (2012) has proposed several species as potential live diets for shrimp larvae
based on their specific characteristics of being mass producible, storable, high in
nutritional value, and capable to be enriched with specific fatty acids. Depending on the
species of nematodes, individual sizes range between 150-2000 μm in length and 15-100
μm in diameter, which puts the smaller individuals in the size class of rotifers and the
larger ones in the size class of Artemia (Sautter et al., 2007).
Furthermore, large scale mass production methods of a specific nematode species,

Panagrellus redivivus, have already been shown by Focken et al. (2006), which is
paramount for a successful application as a commercial Artemia substitute and would
allow implementation in aquaculture productions without supply shortages. Recently,
research has focused on another mass producible nematode species, Panagrolaimus spp.
(strain NFS- 24-5), for its ability to survive a specific dehydration level that makes it
eligible for long term storage, similar to Artemia cysts (Honnens et al., 2013). To
reactivate the nematodes from their desiccated state, simple rehydration for one hour is
needed, after which they are also able to survive extended periods in saline water. This
species also synthesizes two essential fatty acids, eicosapentaenoic acid (EPA) and
arachidonic acid (ARA), and can be enriched with docosahexaenoic acid (DHA)
(Honnens et al., 2014), making it a favorable nutritional diet for shrimp larvae culture.
Recently, Seychelles et al. (2018) and Seychelles et al. (2017) have already successfully
tested Panagrolaimus sp. as a live feed for P. vannamei during the hatchery phase from
the Zoea 2 stage until PL6 with promising results. Several authors conclude that, when
they are provided in sufficient amounts, nematodes are an effective and favorable live
food for the rearing of many crustacean species, yielding excellent survival and growth
performances that make them suitable as a replacement of Artemia (Brüggemann, 2012;
Focken et al., 2006; Seychelles et al., 2018, 2017). But further feeding trials during
different PL stages and under various rearing conditions are needed in order to
thoroughly evaluate the capability of Panagrolaimus sp. (strain NFS-24-5) to be a
complete substitute of live Artemia.
14
1.9. Objectives and Goals
The objective of this work is to evaluate an artificial Artemia diet and a desiccation-
tolerant nematode species to replace live Artemia nauplii, supplemented during the
nursery phase of P. vannamei from PL12 to PL 30 in a zero-water exchange biofloc rearing
system. The results of this feeding trial may potentially lead to increased sustainability
of shrimp farming through alternative feeds that are independent of finite Artemia
resources. In addition, simplifying the feeding processes in shrimp hatcheries may
eventually increase the availability of PL and benefit sustainable shrimp aquaculture
operations.
15
2. Material and Methods
2.1. Preparation of the Experimental Facility
The experiment was carried out in March 2018 at the Laboratório de Camarões Marinhos
(LCM) of the Universidade Federal de Santa Catarina (UFSC) in Barra da Lagoa,
Florianopolis (Santa Catarina, Brazil). Prior to the arrival of the PL from the shrimp
hatching company Aquatec LTDA (Rio Grande do Norte, Canguaratema, Brazil), the
experimental room and all of the equipment was prepared. Sixteen experimental units
(EUs) with a filling capacity of 60 L and consisting of semi-cylindrical blue plastic tanks,
polyvinyl-chloride (PVC) pipes to supply constant aeration in the bottom of the tanks,
and immersion heaters were set up in a wet laboratory of the LCM (Figure 1). Each tank
was evenly equipped with six vertical PVC frames (0.16 m² each) spreading an artificial
surface of Needlona®, a synthetic filter media fabric. The surface area of six Needlona®
frames was equivalent to 100% of the inside surface area of the semi-cylindrical tanks
(0.89 m2), thus doubling the total surface area within each tank. Previous studies have
shown that an artificial substrate of Needlona® is capable to reduce the accumulation of
suspended solids while ensuring high survivals of PL reared in a biofloc nursery system
(Costa Rezende et al., 2018). The air leading PVC pipes ran along the bottom of the tanks
to provide a constant flow of air to assure efficient stirring of the biofloc solution and to
increase the DO (Figure 1 A). The immersion heaters were connected to electrical
contacts hanging from the ceiling above the tanks and were configured to maintain a
constant water temperature of 28-30 °C. The room was also equipped with three
additional radiators to help maintain high temperatures within the experimental room.
One day before the start of the feeding trial, all equipment was thoroughly cleaned,
disinfected with bleach, and again tested for orderly functioning. In addition, the water
pipes of the facility were properly flushed and cleaned by purging a sponge through
them.
16
Figure 1: A: Experimental Unit (EU); B: Row of EUs filled with biofloc water; C: Overview of the wet
laboratory room.
Two days before the arrival of the PL via air transportation, a 4000-L semi-cylindrical
holding tank was set up with a bottom PVC tube for aeration and two flow-through
heaters set at 28 °C (Figure 2 A). The tank was filled halfway with mature biofloc water
from a grow-out pond at the LCM. The other half was filled up with filtered seawater at
33 ppt. The PL arrived late in the evening on the 9th of March 2018 at the Airport of
Florianopolis. They were immediately picked up with a transporter and driven to the
LCM. The total amount of approximately 165000 PL was packaged in 21 carton boxes
containing plastic bags filled with seawater under a pure oxygen atmosphere. The plastic
bags were immediately opened upon arrival to the lab and poured into a specialized
bucket for concentration. The overflow of the bucket was covered by a 500 µm mesh to
keep the PL from washing out (Figure 2 B). It was constantly flushed to exchange the
transport water with clean and filtered seawater. After emptying 7 plastic bags and
washing the PL with seawater, they were carefully transferred to the prepared holding
tank. Another batch of 7 plastic bags was processed again until there were no bags left.
The PL were kept in the holding tank for the next 4 days to allow for acclimation. During
17
this time they were hand-fed 8 times per day (8h00, 10h00, 12h00, 14h00, 16h00, 18h00,
21h00, and 24h00) with a dry feed (DF) (INVE). According to the feed producer’s
recommendations, ±1 g of DF was given at each feeding time.
Figure 2: A: Holding Tank; B: Bucket with meshed overflow.
During acclimation, EUs were filled with 50% water coming from a mature biofloc grow-
out tank and 50% filtered seawater at 33 ppt. In the morning of the 14th of March 2018,
PL were flushed out through the bottom drain of the holding tank, concentrated and
transferred to a 1000 L mixing tank, filled with fresh seawater. Very strong aeration was
applied to achieve a homogenous mixing of the PL inside the tank. A 1-L container was
used to quickly determine the density in PL/L. The container was emptied gradually
over a sieve to count the number of PL retained. All counted PL were quickly returned
to the tank by submerging the sieve. The total number of PL/L was calculated by the
sum of all counted shrimp. This technique was repeated in three replicates to increase
the accuracy of the PL density. Water from the 1000 L mixing tank was reduced to reach
a density of 900 PL/L. While the mixing tank was under very strong aeration, 3600 PL
contained in 4 scoops of 1-L were concentrated onto a sieve and transferred to each EU.
Four EUs were randomly assigned to one of the four dietary treatment groups (Control
[C], Artificial Artemia [AA], Nematodes [N], and Live Artemia [LA]).
18
2.2. Feeding Trial
The feeding trial started on the 15th of March 2018, one day after the PL have been
transferred to the EUs. For the entire experiment, PL were hand-fed 8 times per day
(8h00, 10h00, 12h00, 14h00, 16h00, 18h00, 21h00, and 24h00), whereas at 12h00 and at
18h00 the different treatment groups received their respective special diet. The control
group [C] received the normal DF in the same volume as it was given at all other feeding
times. The [AA] group received a special dry feed composed of extracted and processed
Artemia cyst content. Both feed types, the DF by INVE and the artificial Artemia (AA)
DF, were available in different pellet sizes, called “PL” and “XL” for the DF and
“Standard” and “Large” for the AA. According to the producer’s recommendations,
depending on the PL age, the smaller, larger, or a mix of both pellet sizes, was given. The
live diet groups received a freshly prepared live feed in a quantity based on a study by
Suita et al. (2016), who proposed 70 Artemia individuals per PL per day. The [LA] group
received this quantity of Artemia nauplii, whereas the [N] group received an equivalent
amount in nematode dry weight of the species Panagrolaimus sp. (NFS 24-5). The precise
amount of DF and special feed that was calculated and administered at each time during
the experiment can be seen in Table 1, and the preparation and properties of each type
of feed are described below in more detail. In order to maintain a stable biofloc
formulation with good nitrogen control, dextrose was added to increase the C:N ratio
and calcium hydroxide was added to increase alkalinity. The added amounts were
determined depending on the results from the water analysis for TAN, nitrite, and
alkalinity (Table 2).
19
Table 1: Feeding schedule.
Day 1- Day 5 (PL11-PL14) *
Feeding Times
Treatment 8h00 10h00 12h00 14h00 16h00 18h00 21h00 24h00
Control [C] 0.28g DF 0.28g DF 0.28g DF 0.28g DF 0.28g DF 0.28g DF 0.28g DF 0.28g DF
Artificial Artemia
0.28g DF 0.28g DF 0.57g AA 0.28g DF 0.28g DF 0.57g AA 0.28g DF 0.28g DF
[AA]
Nematodes [N] 0.28g DF 0.28g DF 0.22g N 0.28g DF 0.28g DF 0.22g N 0.28g DF 0.28g DF
Live Artemia [LA] 0.28g DF 0.28g DF 126000 LA 0.28g DF 0.28g DF 126000 LA 0.28g DF 0.28g DF
Day 6 – Day 9 (PL15 –PL18) **

Feeding Times
Artificial Artemia
[AA]
Day 10 – Day 13 (PL19 - PL22) **

Feeding Times
Artificial Artemia
[AA]
Day 14 – Day 21 (PL23 – PL30) **

Feeding Times
Artificial Artemia
[AA]
* From Day 1 to Day 5, DF was given as a 1:1 mix of the sizes “PL” and “XL”; and AA was given as a 1:1
mix of the sizes “Standard” and “Large”.
** Starting from Day 6, DF was given only in the feed size “XL”, and AA was given only in the size
“Large”
20
Table 2: Amount of diet, dextrose, and calcium hydroxide added to all groups at each day.
Day *
Input
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Diet (g) 2.2 2.2 2.2 2.2 2.2 2.9 2.9 2.9 2.9 3.6 3.6 3.6 3.6 4.9 4.9 4.9 4.9 4.9 4.9 4.9
Dextrose (g) - - - - - - - - - - - - - - 2.5 2.0 1.0 1.0 1.0 1.0
Calcium
- - - - 0.6 0.6 0.6 1.1 1.1 1.1 1.1 0.9 0.9 0.9 0.5 0.5 0.5 0.5 0.5 0.5
Hydroxide (g)
* Starting at day 16, the feed was decreased by 50% due to deteriorating water quality.
2.3. Feed Preparation

2.3.1. Preparation of live Artemia
On every day, live Artemia nauplii were prepared for hatching 24 hours before they were
used as feed. Based on the recommendations in a study by Suita et al. (2016), it was
determined that during the nursery phase from PL1 to PL30, each PL should receive
approximately 70 individuals of Artemia nauplii as feed per day. Thus, each tank of 3600
PL should receive 252000 nauplii per day, so half that amount per special feeding. Since
four tanks received the LA treatment, approximately 1.0 - 1.5 million nauplii, depending
on the experimental phase, were needed each day (Table 1). Prior conducted tests of
hatching Artemia cysts (Artemia High 5, INVE) in this facility and with the available
equipment have shown results with high fluctuations regarding the quantity of
produced nauplii. On average, only between 75000 and 100000 nauplii were successfully
hatched per gram of Artemia cysts. Therefore, to be on the safe side, 20 g of cysts were
prepared for hatching each day, expected to be yielding an excess of 1 million Artemia
nauplii per day. This volume of cysts was raised to 30 g during the later stages of the
feeding trial when more nauplii were needed. Starting at 11h00 in the morning before
the first day of the experiment, 20 g of Artemia cysts were put in 10 L of water at a salinity
of 15-25 ppt. The solution was filled into an Artemia hatching container with an air
supply attached at the bottom (Figure 3). The aeration was set up strong enough to
sufficiently mix the solution without spilling it out of the container. A strong light source
21
emitting approximately 2000 lux was placed just above the water surface and an
immersion heater was inserted in the tank and set to 29 °C. Twenty-four hours later, the
light and aeration were switched off and the heater was removed. A lid was placed above
the container so that the hatched Artemia would swim to the transparent bottom of the
tank, now the only source of light. After half an hour, the water and the hatched Artemia
nauplii were carefully drained from the bottom of the tank into a bucket through the
disconnected aeration pipe, until only the empty shells that were floating atop were left
in the hatching tank. This process of shell and nauplii separation was followed by
removing any remaining floating Artemia shells with a plastic pipette. By filtration
through a 100 µm sieve, the Artemia nauplii were concentrated and then washed into a
beaker with 1000 mL of seawater at 33 ppt to have a known volume for the following
quantity calculations. A small aeration stone, connected to an air delivering system, was
added to ensure sufficient oxygen supply. An aliquot of 1 mL of the Artemia solution was
diluted in 1000 mL of seawater. The total quantity of Artemia was estimated by counting
nauplii contained in 10 samples of 1 mL, using a glass pipette. The calculation was
extrapolated to the concentrated solution of Artemia nauplii to deliver the quantity
required in each EU of LA dietary treatment. The remainder of the hatched Artemia were
stored in a beaker with sufficient aeration to be used as feed for the second special
feeding at 18h00. Any excess of produced Artemia nauplii was frozen inside small plastic
cups and marked with the estimated quantity of individuals inside. These units of frozen
Artemia nauplii were used as backup feed in case the hatching process on a given day
would fail or yield too little live nauplii.
Figure 3: Diagram of Artemia hatching tank.
22
2.3.2. Preparation of Nematodes
The nematode diet was composed of pure dehydrated Panagrolaimus sp. (NFS 24-5)
provided by the company E-nema GmbH (Schwentinental, Germany). Since the
nematodes are not uniform in size but consist of small individuals (150-800 µm length)
and large individuals (800-1300 µm length) it is more practical to estimate the needed
amount equivalent to the LA in weight rather than density. One Artemia nauplius is
estimated to have a dry weight of 1.6 ± 0.1 µg (Farhadian et al., 2009). Thus, the amount
of dehydrated nematodes equivalent to 70 Artemia nauplii is 112 µg. To compensate the
10% of water still existent in the dehydrated state, 123 µg dry weight of nematodes was
used per PL per day. The required quantity of dehydrated nematodes needed for each
day (Table 1) was weighed using a digital scale and put in a glass beaker. They were
rehydrated in 24 mL of freshwater every day 1-hour prior to feeding. The solution was
then thoroughly mixed until it became completely homogeneous. After 30 to 60 minutes
the nematodes were sufficiently hydrated to awaken from their desiccated state and
began to show their typical sinuous movements, which could be observed under a
binocular microscope. At each special feeding time, 3 mL of the nematode solution was
administered to each tank of the [N] group with a plastic pipette. After the 12h00 feeding,
the remaining half of the daily prepared nematode solution was kept in a fridge at 7 °C
until the evening feeding at 18h00.
2.3.3. Preparation of dry feed and artificial Artemia
The DF used for this feeding trial was manufactured by INVE (Thailand) Ltd. (Nong
Lum, Wachirabarami, Thailand) and consisted of the products INVE EPAC Black PL and
INVE EPAC Black XL, differing only in their particle size. The smaller particle size
product, INVE EPAC Black PL, was only used during the first 5 days, from PL12 – PL17,
mixed in a 1:1 ratio with the larger product XL. Both products had the same inclusion
levels of marine protein, plant protein, pigments, marine oils, yeast extract, minerals,
and antioxidants. The composition of both feeds was also identical, with 45% protein,
23
7% lipid, 3% fiber, and 10% moisture content (INVE Technologies, 2018). The artificial
Artemia (AA) supplement product contained an extract of Artemia cysts and other
ingredients, such as fish gelatin, squid meal, hydrogenated vegetable fat, soy lecithin,
and microalgae. The diet composition was 51% protein, 13.6% lipid, 8% ash, 3.5% fiber
and 8% moisture. This product also came in two particle sizes, called Standard and
Large, where the smaller particle size (Standard) was delivered during the first 5 days in
the same way as indicated for DF. All specifications regarding ingredients were taken
directly from publications provided by the producers. The required quantities of DF and
AA feed (Table 1) needed for each day were weighed in small 50 mL plastic cups. When
all cups were prepared, they were administered simultaneously to the different
treatment groups at the designated time.
2.4. Water Quality Analysis
The temperature and the DO of each EU were measured daily with a YSI 55 device (YSI
Incorporated, Yellow Springs, OH, USA) and recorded in the morning at 8:00 and in the
evening at 18:00. Twice per week 200 mL water samples of each EU were taken in the
morning at 8h00 and analyzed for alkalinity (APHA 2005-2320 B), total ammonia
nitrogen (TAN) a nitrite (APHA, 2005), salinity (YSI 30, YSI Incorporated, Yellow
Springs, OH, USA), and pH (YSI 100, YSI Incorporated, Yellow Springs, OH, USA). Total
suspended solids (TSS) and volatile suspended solids (VSS) (APHA 2005-2040 D and
2005-2540 E) were measured using 0.6 µm glass fiber micro-filters (GF-6, Macherey-
Nagel, Düren, Germany). TAN and nitrite analysis were carried out following a protocol
from Strickland and Parsons (1972) and the guidelines of APHA (2005). All water analysis
procedures were carried out by professional technical staff in a specialized laboratory of
the LCM.
24
2.5. Salinity Stress Test
One day before the end of the experiment, a salinity stress test was performed to
determine the PL’s ability to survive an osmotic shock. Thirty PL from each EU were
randomly selected with a 500 µm sieve and placed in a 500 mL plastic cup containing
freshwater at the same temperature as the EU (~29 °C). To minimize handling stress,
the whole sieve containing the PL was placed inside the freshwater cup. After 45
minutes, the sieve was removed and placed in a cup of seawater at 33 ppt and ~29 °C.
Another 45 minutes later, all surviving and dead PL were counted. A PL was determined
to be dead when it did not show any movement after a stimulus with a plastic probe.
2.6. Final Sampling
On the final day of the experiment (Day 20, PL31), the larvae from each tank were
collected by siphoning the water through a 500 µm sieve. Each individual sieve and the
PL within were carefully blotted with a piece of paper to remove excess water and were
then weighed together. Thereafter, the sieves, freed of the PL, were dried in an oven at
60 °C for 2 hours and then weighed again. The individual weight of each sieve was
subtracted from the total (sieve and PL) to obtain the total wet weight of all PL per tank.
A smaller quantity of PL from each tank was weighed separately and subsequently
counted in order to estimate the wet weight per larvae. The counted PL were stored and
preserved in formaldehyde. All other remaining PL were stored in plastic bags and
frozen at -20 ºC for further body composition analysis. At a later date, the formaldehyde-
preserved PL were placed on a white paper and measured from rostrum to tail to obtain
the mean total length of each treatment group. The relative growth rate (RGR) was
calculated to analyze potential differences in growth between the groups with:
RGR (%/day) = (eg−1) × 100, (1)
25
where g = (ln Wt - ln W0) × t−1, Wt and W0 are the final and initial wet weights,
respectively, and t is the duration of the trial in days (Aragao et al., 2007). After the
finalization of the experiment, the frozen PL were transferred to a commercial
laboratory where a body composition analysis for ash, lipid and protein contents was
performed.
2.7. Statistical Analysis
All data was analyzed using the statistical software program RStudio (Version 1.1.453 –
© 2009-2018 RStudio, Inc.). The Shapiro-Wilk test was used to test the obtained data for
normal distribution (Royston, 1995) and the Levene’s test determined the homogeneity
of variance (Fox, 2016). When the data was found to be normally distributed and with
homogeneous variance, a one-way analysis of variance (ANOVA) (Chambers & Hastie,
1992), followed by Tukey’s honest significant differences (HSD) post-hoc test (Yandell,
1997) was performed. The water parameters measured over time were analyzed using a
repeated measures ANOVA with the treatment group as the main factor and the
sampling day as the additional factor. Any data set that was found to be not normally
distributed and/or without homogenous variance, was analyzed using the non-
parametric Kruskal-Wallis Rank Sum test (Hollander et al., 2015), followed by Dunn’s
test (Dunn, 1964). All significant differences were determined at a confidence level of a
= 0.05.
26
3. Results
3.1. Water Quality Parameters

3.1.1. Temperature and Salinity
Temperature and salinity did not show any significant differences among treatments
from day 1 until day 20, the end of the experiment (Figure 4 andFigure 5). Utilizing
electric submergible heaters, the mean temperature was kept between 28 °C and 29.5 °C
at all times. The maximum recorded temperature was 29.2 ± 0.3 °C for the [N] group at
day 16, while the lowest temperature of 28.3 ± 0.3 °C was recorded in the [AA] group at
day 1. There were some minor fluctuations in salinity throughout the feeding trial, but
the rearing water never reached more than 36 ppt in any tank. The highest salinity of 35
± 0.4 ppt was measured on day 5 in the [C] group and the lowest salinity of 33.1 ± 0.2 ppt
occurred at the start of the experiment and was equal in all treatments. Whenever an
increase in salinity was observed in a specific tank, the adequate amount of disinfected
freshwater was added to counter a rising salinity.
Figure 4: Temperature in each treatment along the experiment. Values are means ± SD (n=4). The
absence of letters indicates no statistically significant differences on the given day.
27
Figure 5: Salinity of the four treatments on each sampling day. Values are means ± SD (n=4). The
absence of letters indicates no statistically significant differences on the given day.
3.1.2. Dissolved Oxygen
For the most part of the experiment, the DO levels were similar among the four
treatments. But during the final stages, after day 15, some statistically significant
differences were observed. The initial DO levels in all treatments was 7.14 ± 0.02mg/L
and DO levels slowly dropped until they reached their lowest point of 6.8 ± 0.22 mg/L
on day 15 in the [C] group, after which they slowly started to rise again. On day 18, the
DO level of the [LA] group was significantly lower (6.78 ± 0.09 mg/L) than that of the
[AA] group (6.99 ± 0.07 mg/L) and the [N] group (6.96 ± 0.05 mg/L). There was no
significant difference to the [C] group (6.91 ± 0.07 mg/L). On day 19, the following day,
no statistically significant differences were observed among groups, while on the final
day, day 20, again the [LA] group (7.02 ± 0.06 mg/L) had significantly lower DO than
the [AA] group (7.18 ± 0.09 mg/L). But neither group, [LA] nor [AA], had a significantly
different DO level compared to the [N] group (7.13 ± 0.07 mg/L) or the [C] group (7.06
± 0.04 mg/L), as shown in Figure 6.
28
Figure 6: Dissolved oxygen in each treatment along the experiment. Values are means ± SD (n=4). The
absence of letters indicates no statistically significant differences on the given day. Different letters
represent statistically significant differences on the given day (p < 0.05).
3.1.3. Alkalinity
The water alkalinity analysis at the start of the experiment returned a starting value of
145.0 ± 0.0 mg/L in all treatments. At the first two sampling points, on day 5 and day 8,
the alkalinity dropped to 120.0 ± 4.0 mg/L and then to 100.0 ± 3.6 mg/L, respectively, in
all treatments. In order to increase the alkalinity in the water, calcium hydroxide was
added to each tank at a rate of 20% of total feed given per day. The daily ratio was given
in two servings at 11h00 and 15h00 starting on the afternoon of day 5. Since the alkalinity
still decreased until day 8, the calcium hydroxide dosage was doubled to 40% of the total
feed given per day. An overview of the exact quantities of calcium hydroxide added each
day can be found in Table 2. At day 12, the alkalinity started rising slowly and was now
again above 100.0 ± 5.5 mg/L in all treatments. Until this point in the experiment, there
were no statistically significant differences among the groups. On the next sampling
point on day 15, the alkalinity had risen to 130.0 ± 4.2 mg/L in all treatments except for
29
the [N] group, which showed a significantly higher value of 149.0 ± 6.0 mg/L. The
alkalinity on the final day of the experiment, day 19, had decreased slightly in all groups
to 123.7 ± 2.5 mg/L, whereas again, the [N] group had the highest and significantly
different value of 142.0 ± 5.2 mg/L (Figure 7).
Figure 7: Alkalinity of the four treatments on each sampling day. Values are means ± SD (n=4). The
absence of letters indicates no statistically significant differences. Different letters represent
statistically significant differences among the treatments on the given day (p < 0.05).
3.1.4. pH
The pH of the biofloc water started out slightly basic in all treatments (8.21 ± 0.04) and
decreased slowly until day 8 to 7.74 ± 0.07. Towards the end of the experiment, the pH
in the [N] group was significantly higher on day 15 (8.00 ± 0.02) and day 19 (7.98 ± 0.03)
than in the other three groups, where the pH was below 7.92 ± 0.03 and 7.91 ± 0.02,
respectively (Figure 8). To determine a potential correlation between alkalinity and pH,
a Spearman rank test was performed using the complete data sets of these two
parameters. A positive correlation with a correlation coefficient of r = 0.56 was
determined (p < 0.001; Figure 9).
30
Figure 8: The pH level of the four treatments on each sampling day. Values are means ± SD (n=4). The
absence of letters indicates no statistically significant differences. Different letters represent
statistically significant differences among the treatments on the given day on the given day (p < 0.05).
Figure 9: Pearson correlation of alkalinity and pH with data from all groups on all sampling days.
31
3.1.5. Total Suspended Solids
The water analysis at the start of the experiment accounted for 303.0 ± 36.5 mg/L of total
suspended solids (TSS) in the initial biofloc water. During the following sampling days,
several statistically significant differences were observed (Figure 10). Most strikingly, on
the final two sampling days, day 15 and day 19, a significant difference between the [AA]
and [C] group and the [N] and [LA] group were noticed. On day 15, the [AA] group (437.8
± 34.6 mg/L) and the [C] group (431.5 ± 23.9 mg/L) had significantly higher TSS values
than the [N] group (264.5 ± 38.6 mg/L) and the [LA] group (260.0 ± 62.8 mg/L).
Similarly, although with a smaller difference in magnitude, the same pattern was
observed on the measurements on day 19, where the [AA] (423.0 ± 12.3 mg/L) and [C]
(416.3 ± 16.7 mg/L) groups had significantly higher values of TSS than the [N] (344.0 ±
17.8 mg/L) and [LA] (340.0 ± 40.5 mg/L) groups.
Figure 10: Total suspended solids of the four treatments on each sampling day. Values are means ± SD
(n=4). The absence of letters indicates no statistically significant differences. Different letters represent
32
3.1.6. Volatile Suspended Solids
The volatile suspended solids (VSS) analysis showed a very similar pattern compared to
the TSS. All treatments had the same initial value of 108.8 ± 16.0 mg/L VSS at the start
of the experiment. During the course of the feeding trial, several statistically significant
differences were determined among the groups, and again, during the final stage of the
experiment, on day 15, a clearly significant difference between the control and the [AA]
groups and the two life feed groups, [N] and [LA], was observed (Figure 11). On the last
sampling, day 15, the [AA] (171.8 ± 26.6 mg/L) and the control (165.8 ± 10.7 mg/L) groups
had significantly higher VSS values than the [N] (97.3 ± 8.5 mg/L) and the [LA] (89.0 ±
13.2 mg/L) groups. Looking at both parameters of suspended solids, TSS and VSS, during
the latter phase of the experiment, and especially on the final day of sampling (day 19
and day 15, respectively), the inert diet groups (control and [AA]) always had statistically
significant higher values than the groups fed with live diets ([N] and [LA]).
Figure 11: Volatile suspended solids of the four treatments on each sampling day. Values are means ±
SD (n=4). The absence of letters indicates no statistically significant differences. Different letters
represent statistically significant differences among the treatments on the given day (p < 0.05).
33
3.1.7. Total Ammonia Nitrogen
The total ammonia nitrogen (TAN) concentrations at the beginning of the experiment
started relatively low with 0.25 ± 0.03 mg/L in all treatments but began fluctuating
during the course of the trial (Figure 12). On sampling day 5, the [AA] group (0.35 ± 0.07
mg/L) had a slightly significant higher TAN concentration than the [N] (0.25 ± 0.02
mg/L) and [LA] (0.23 ± 0.01 mg/L) groups. No statistically significant differences were
found between either group and the control group (0.27 ± 0.02 mg/L) on that day. On
all other sampling days, no significant differences among the treatments could be
determined. Overall, a decrease in the mean TAN levels towards the end of the
experiment was observed. The highest measured peak of TAN during the feeding trial
occurred on day 8 in the [LA] group with 0.44 ± 0.25 mg/L.
Figure 12: Total ammonia nitrogen values of the four treatments on each sampling day. Values are
means ± SD (n=4). The absence of letters indicates no statistically significant differences. Different
letters represent statistically significant differences among the treatments on the given day (p < 0.05).
34
3.1.8. Nitrite
From the start of the experiment, until past day 8, almost no nitrite was detected in the
biofloc system of all treatments (< 1.4 ± 0.2 mg/L). Starting from day 12, nitrite levels
started to rise relatively quick, until they reached critical levels on the final sampling,
day 19. On day 12, 15, and 19, statistically significant differences among the groups were
observed, whereas, on all of these days, the [N] group had significantly lower nitrite than
all other groups (Figure 13). Nitrite peaked on the last day, with the highest value in the
[LA] group (30.0 ± 1.7 mg/L) followed by [AA] (29.2 ± 1.5 mg/L) and the control group
(26.1 ± 2.5 mg/L). Compared to the [LA] and [AA] group, the [N] group had a significantly
lower nitrite level (23.6 ± 1.6 mg/L) on that day, while the [C] group was not significantly
different to any other group.
Figure 13: Nitrite values of the four treatments on each sampling day. Values are means ± SD (n=4).
The absence of letters indicates no statistically significant differences. Different letters represent
35
3.2. Growth Performance
3.2.1. Survival
The results of the final survival showed that PL fed with live diet supplements, the
groups [N] and [LA], had a statistically significant higher survival at the end of the
experiment compared to the two groups fed only with an inert diet, [C] and [AA]. The
[N] group had a final survival of 94 ± 6% and the [LA] group of 91 ± 7%, whereas the [C]
and [AA] group only had a final survival of 51 ± 10% and 53 ± 11%, respectively (Figure
14).
Figure 14: Final survival of P.vannamei post larvae from each treatment at the end of the experiment
(n=4). Box = 25th and 75th percentile, Bars = min and max values, thick line = median value. Different
letters indicate statistically significant differences, which were determined by a one-way ANOVA (p <
0.0001), followed by Tukey’s HSD (p < 0.05).
36
3.2.2. Growth
The mean wet weight per larvae, determined right after the completion of the
experiment, returned a statistically significant difference between the two inert diet
groups and the two live diet groups (Table 3). The mean wet weight of the larvae from
the [C] (35 ± 10 mg) and [AA] (35 ± 6 mg) groups was slightly, but significantly higher
than the weight of the larvae from the [N] (23 ± 5 mg) and LA (23 ± 5 mg) groups. Similar
to the wet weight results, the mean individual length results returned significant
differences between the inert diet and the live diet groups (Table 3). The [C] (17.3 ± 5.0
mm) and [AA] (17.1 ± 5.2 mm) groups had significantly longer PL mean length than the
[N] (15.8 ± 3.9 mm) and [LA] (15.8 ± 3.2 mm) groups.
Table 3: Growth of P. vannamei larvae from different treatments at the end of the experiment.
RGR: relative growth rate.
Wet Weight Total Length RGR

Groups
(mg) (mm) (% day -1)
Control [C] 35 ± 10 a 17.3 ± 5.0 a 15.2 ± 1.5 a
Artificial Artemia [AA] 35 ± 5.7 a 17.1 ± 5.1 a 15.3 ± 1.0 a
Nematodes [N] 22.5 ± 5 b 15.8 ± 3.9 b 12.8 ± 1.2 b
Live Artemia [LA] 22.5 ± 5 b 15.8 ± 3.2 b 12.8 ± 1.2 b
* Values are means ± SD (n=4 for all except total length: C: n = 175; AA: n = 189; N: n = 222; LA: n =
246). Different superscript letters within the column indicate significant differences among groups (p
< 0.05). Initial wet weight was 2 ± 0 mg.
The calculation and analysis of the relative growth rate returned statistically significant
differences between the inert diet and the live diet groups. [C] and [AA] treatments had
an RGR of 15.2 ± 1.5 and 15.3 ± 1.0%/day, respectively, whereas both treatments [N] and
[LA] only had an RGR of 12.8 ± 1.2%/day (Table 3).
37
3.2.3. Body Composition
The analysis of the body composition for lipids, ash, and protein content was performed
by a commercial laboratory (LABCAL) and the results are presented in Figure 15.
Unfortunately, there was not enough sample tissue available to analyze replicates per
group, thus, no standard deviation nor statistical significance could be calculated. PL
from all treatments had around 10-12% protein, 4-5% ash, and 1% lipid content.
Figure 15: Post larvae body composition at the end of the experiment. Due to the lack of replicates, no
statistical analysis could be performed.
38
3.2.4. Salinity Stress Test
The salinity stress test did not return any significant differences among the treatments.
All groups had a high survival between 88 ± 6% for the [N] group and 94 ± 6% for the
[LA] group (Figure 16).
Figure 16: Survival of P.vannamei post larvae from each treatment (n=30) after the salinity stress test.
Box = 25th and 75th percentile, Bars = min and max values, thick line = median value. The absence of
letters indicates no statistically significant differences among the treatments.
39
4. Discussion
4.1. Water Quality Parameters
The temperature and salinity levels were kept relatively constant around 29 °C and 33-
35 ppt for the entire experiment and no significant differences were observed among the
groups. The species P. vannamei naturally occurs in oceanic habitats with all year
temperatures above 25 °C (Dugassa & Gaetan, 2018), hence, the temperature and salinity
of this experiment can be considered optimal. During all times DO concentrations were
maintained well above the limits considered adequate for aquaculture (> 5 mg/L) (Boyd
& Gautier, 2000). Thus, although statistically significant differences were observed
among the groups at days 18 and 20, no effect of DO on the larval growth is expected.
The alkalinity decreased in all treatments despite the addition of calcium hydroxide
before it slowly increased again towards the end of the experiment. The pH level in the
water followed a very similar course to the alkalinity and this parallel behaviour was
expected and confirmed with a positive correlation by the Spearman rank test. The
alkalinity of water is very closely related to pH, and generally, pH tends to increase as
alkalinity increases and vice versa (Boyd, 2000). Both parameters can have an impact
on the bacterial composition of the biofloc and subsequently on the nitrogen cycle in
the water. The nitrification of ammonia to nitrate is a two-step conversion performed
by different genera of chemoautotrophic bacteria. In a first step ammonia oxidizing
bacteria (AOB) of the genus Nitrosomonas metabolize ammonia to nitrite and in a
second reaction, nitrite is oxidized to nitrate by nitrite oxidizing bacteria (NOB) of the
genus Nitrobacter (Chen et al., 2006). The optimum pH for the growth of nitrifying
bacteria varies widely depending on the species, but the ideal pH for the nitrification
process is between 7.2 to 9.0 for Nitrosomonas and 7.2 to 8.8 for Nitrobacter (Chen et
al., 2006; Villaverde et al., 1997). At day 8 of the experiment, the mean pH in all
treatments dropped to 7.8 ± 0.1, which was closer to the lower pH limit proposed by
Villaverde et al. (1997). The authors also argue that AOB have faster growth than NOB
and the latter also show a higher sensitivity to pH. It is therefore likely that selective
40
inhibition of NOB occurred, which subsequently resulted in the observed accumulation
of nitrite starting after day 8 of the experiment. Both steps of the nitrification process
also consume considerable amounts of alkalinity, as carbonate and bicarbonate are
essential nutrients for nitrifying bacteria (Chen et al., 2006). A reduction in alkalinity
leads to a decreased buffering capability of the rearing water resulting in acidification
and a reduced pH. This negative feedback loop could have inhibited the nitrification
process further and should have been avoided by timely addition of calcium hydroxide
or other carbonate sources.
The authors Ebeling et al. (2006) and Hargreaves (2006) suggested that different
pathways of nitrogen removal are to some degree present at the same time in biofloc
systems. The three general ways to recycle nitrogen are photoautotrophic uptake by
algae, autotrophic bacterial conversion from ammonia to nitrite and nitrate, and
heterotrophic bacterial assimilation of nitrogen into microbial biomass. Vinatea et al.
(2010) argue that the latter two bacterial processes play the dominant role in high
density and indoor nursery systems, as photosynthesis by phytoplankton is minimal due
to high turbidity and limited natural light. According to Avnimelech (2006),
heterotrophic assimilation and autotrophic nitrification can take place at the same time
on the surface of a mature biofloc. In order to support both bacterial processes of
nitrogen reduction, the alkalinity levels should be kept between 100-150 mg/L at all times
(Ebeling et al., 2006), which was only achieved after day 12 of the experiment. Chen et
al. (2006) recommend even higher alkalinity concentrations of above 200 mg/L in zero
water exchange systems, since a stratification of alkalinity and pH in the biofilm on the
substrate surface may occur. Thus, the temporary drop in alkalinity and pH may have
hampered the growth of the NOB and manipulated the microbial community in the
biofloc towards an unfavourable composition regarding nitrite control. This assumption
is in accordance with findings by Furtado et al. (2015) who studied the effects of different
alkalinity levels for the rearing of P. vannamei in BFT and concluded that low alkalinity
negatively effects NOB. In this experiment the nitrite levels started to rise in all groups
beginning on day 12, suggesting an overall reduced alkalinity supply. In contrast, the
TAN levels were continuously decreasing in all treatments toward the end of the
experiment. This observation further indicates the presence of a chemoautotrophic
41
bacterial nitrogen conversion from TAN to nitrite, without the subsequent step from
nitrite to nitrate. In a pure heterotrophic system, there should have been no nitrite build
up at all, since the ammonia nitrogen would have been assimilated directly into bacterial
biomass (Ebeling et al., 2006).
On the final two sampling days (15 and 19 days of experiment) a clear statistically
significant difference was observed, where the [N] group had higher alkalinity and pH
then all other groups. Consequently, the [N] group had also significantly lower nitrite
levels on these days, suggesting a different level of bacterial nitrogen control present in
the nematode treatment. After day 12, the nitrite continued to increase in all groups until
it reached dangerously high levels between 24-30 mg/L at day 19. In order to not further
deteriorate the water quality by increasing the organic matter, the daily feed input in all
tanks was decreased by 50% from day 16. But nevertheless, a continuous increase in
nitrite was observed. Lin & Chen (2003) estimated that a concentration of 25.7 mg/L of
nitrite nitrogen is the upper safe level limit for rearing P. vannamei PL of approximately
4 g at 35 ppt salinity. In the final days of the experiment, this limit was exceeded, so the
nitrite accumulation may have had a negative effect on the shrimp’s survival and growth.
Especially, since the PL in this experiment were much smaller at the final sampling with
an average of 28.8 ± 8.6 mg and previous studies suggested that penaeid shrimp
tolerance to nitrite increases with age (Lin & Chen, 2003; Ramírez-Rochín et al., 2017).
According to a study by Vinatea et al. (2010), nitrite concentrations close to 9 mg/L may
already cause a significant reduction in the growth rate of shrimp. But since this level
was exceeded only after day 14 in all treatments, differences in survival and growth of PL
among the groups due to elevated nitrite concentrations may not be evident at the final
sampling. The significant lower nitrite levels of the [N] group did not result in better
growth compared to the other groups, as the highest RGR was observed in the [C] and
[AA] groups. Since the highest values of nitrite and the excess of the safe level only
occurred on the last days of the experiment, mortalities that would occur after long-
term exposure should not have affected the final survival of the PL.
TAN in none of the treatments ever exceeded 0.5 mg/L and was constantly well below
the safe levels for P. vannamei in biofloc systems, which are considered to be around 4.0
42
mg/L of TAN at 35 ppt salinity (Boyd & Gautier, 2000; Lin & Chen, 2001). Nitrate, the
final product of the nitrification process, was not monitored during the experiment
because the autotrophic conversion of nitrogen was not expected to have an important
role. But since nitrate is the least toxic form of nitrogen (Romano & Zeng, 2013), it is
assumed that a potential accumulation did not have adverse effects on the PL
performance.
Microbial communities in biofloc are very complex systems, but they can be shifted
towards predominantly heterotrophic bacterial growth by increasing the
carbon:nitrogen ratio. It is possible that this shift did not occur at a sufficiently fast rate
and more rigorous addition of dextrose earlier in the course of this experiment could
have promoted a stronger growth of heterotrophic bacteria (Ebeling et al., 2006). The
specific growth of the microbial community has implications on the formation of the
biofloc, its ability to control nitrogen build-up in the system, and the accumulation of
suspended solids. According to Schveitzer et al. (2013) and Samocha et al. (2007), TSS
levels between 300-600 mg/L are optimal for rearing P. vannamei larvae. Within this
range, the biofloc nitrogen control is most effective while the respiration of the shrimp
is not handicapped. TSS levels in all groups fluctuated between 200-450 mg/L and were
within the desired limits. Hence, a general adverse impact on the shrimp’s growth
performance through TSS is not expected. However, a clear distinction between the
groups fed with inert diets, [C] and [AA], and live diets, [N] and [LA], started to develop
over the course of the experiment. On the last two sampling days, day 15 and day 19, the
live diet groups had statistically significant less TSS than the inert diet groups, indicating
a possible effect of the live diet on the biofloc formation itself. At the last sampling at
day 15, the groups that received a supplement of live Artemia or live nematodes also had
statistically significant differences and approximately 50% lower VSS levels than the
groups that received only DF.
Over the course of the experiment, the live diet groups received approximately 25% less
DF than the inert diet groups. This difference in inert organic material could have caused
divergent developments in the biofloc formulation, leading to greater TSS and VSS
accumulations in the [C] and [AA] treatments. The final sampling revealed significant
43
lower survivals in the inert diet groups, but it remains unclear at what time during the
experiment these mortalities occurred. Hence, the feed input was not adjusted to the
reduced quantity of PL and it is, therefore, possible that overfeeding led to the increase
in TSS and VSS. The exact cause of the mortalities is also unknown, but the most
plausible hypothesis in this scenario is the nitrite increase, which reached levels above
10 mg/L at day 14. Although Lin & Chen (2003) did not report this nitrite level as lethal
toxic to juvenile shrimp, the possibility that smaller shrimp may suffer mortalities
cannot be excluded. But since a clear divergence in the TSS levels between inert and live
diet groups occurred as early as day 12, two days before the nitrite peak, it may be
assumed that the presence of live diets in the biofloc solution somehow reduced the
accumulation of TSS and VSS in the [N] and [LA] groups as well.
Many free-living nematode species are bacterivores and predominantly feed on bacteria,
while some species even occur naturally in biofloc environments (Emerenciano et al.,
2013). If the Panagrolaimus sp. (strain NFS-24-5) survives extended periods of time in
salt water, as other nematode species do (Brüggemann, 2012), they may have been
ingesting bacteria from the biofloc and thereby reducing the extensive accumulation of
VSS and TSS in the [N] group.
Artemia only start feeding once they reach the metanauplii stage, which happens after
6 – 8 h in temperatures above 25°C (Sorgeloos et al., 1998). Metanauplii are passive filter
feeders that will ingest anything small enough, such as microalgae or bacteria
(Fernández, 2001). Since the Artemia nauplii were stored for 6 h until the 18h00 feeding
at ambient temperature (~25 °C), it is possible that they had developed into metanauplii
by the second special feeding. Luo et al. (2017) and Yao et al. (2018) have recently shown
that Artemia can feed very efficiently on the microorganisms in biofloc and thereby
encapsulate valuable nutrients and probiotics. If nematodes and Artemia metanauplii
were able to survive predation by the PL, or if they were administered in excess so that
a population of live diets remained in the rearing water, they may have consumed some
part of the biofloc. This could explain why the TSS and VSS levels in the [LA] and [N]
groups were statistically significant lower than the levels in the inert diet groups.
Sorgeloos et al. (1998) also state that metanauplii are almost transparent, less visible and
44
fast swimming, therefore less acceptable as prey. Nevertheless, P. vannamei PL are very
efficient hunters that feed frequently (Peixoto et al., 2017; Sanudin et al., 2014), and at
least the nematodes have no means to actively avoid predation. Besides, the live diets in
the [N] and [LA] groups are very different, coming from completely separated animal
groups with varying feeding mechanisms, but the reduced TSS levels of both live diet
groups were very similar. The assumption that nematodes and Artemia reduced the
accumulation of suspended solids to the same degree by feeding on the biofloc remains
questionable. But it could be further investigated with an experimental design, in which
the tanks are periodically checked for survivors at certain times after feeding. Also, a gut
analysis of nematodes and Artemia could be performed to estimate the rate of their
biofloc ingestion and ability to reduce TSS and VSS.
According to the feeding regime, the [N] and [LA] group received about 25% less DF
over the course of the experiment, which was supplemented by their specific live diet. If
the provided feed was insufficient, it may be possible that the shrimp in these groups,
in order to avoid starvation, were grazing more intensely on the biofloc and therefore
partially removed it from the system. The final growth results showed that PL from the
live diet groups were slightly but still significantly lighter and shorter than the PL from
the inert diet groups, which would support the assumption of a suboptimal feeding
regime compared to the inert diet groups.
Several authors have investigated the nutritional contribution of biofloc and it was
reported that up to 29% of daily food uptake by P. vannamei could stem from biofloc
material with concurrent improvements of growth rate (Burford et al., 2004;
Emerenciano et al., 2013). But since all groups in this experiment were reared in biofloc
and no quantitative analysis of the biofloc ingestion by the shrimp was done, this
observation cannot be affirmed with this experiment. The interpretation of the water
quality parameters rather points to the assumption that the supplement of live diets to
the feeding regime led to a change of the biofloc composition, which in turn had an
effect on the PL performance. Since both live diets had an impact on the TSS and VSS,
but only the [N] group had significantly different results in nitrite, alkalinity, and pH, it
can also be assumed that there is a disparity between nematodes and live Artemia and
45
their effect on the biofloc system with ensuing implications for the water quality. Further
experiments investigating the impacts of live diets, especially nematodes, on biofloc and
water quality should be designed in order to better understand the complexity of the
processes. A first presumption from this experiment is that the nematode species
Panagrolaimus sp. (strain NFS-24-5) may be beneficial for the water quality in a biofloc
system.
4.2. Larval Performance
The analysis of the final survival of the PL revealed a clear distinction between inert and
live diets with statistically significant differences. The [N] and [LA] groups had nearly
twice the survival rate than the [C] and [AA] groups at the end of the experiment. As
mentioned before, the exact cause of the mortalities and at what moment they occurred
remains unclear. It is also plausible that the PL in the inert diet groups died gradually
over the time of the experiment, rather than at one incident. No dead individuals were
observed during the daily routines or at the final harvest of the PL. This occurrence is
expected, as P. vannamei are omnivorous scavengers with occasional cannibalistic
behaviour, especially during the nursery phase with high metabolic rates (Romano &
Zeng, 2017). Any deceased PL would have been readily consumed by the survivors,
potentially increasing their feed input and growth.
Compared to the low survival rates of the [C] and [AA] groups (51 ± 10% and 53 ± 11%,
respectively), Costa Rezende et al. (2018) reported high survival (91 ± 12%) in a similar
experimental setup with P. vannamei reared from PL5-PL20, fed with inert diet alone
and no observed nitrite peak. The water quality of this experiment, in regard to elevated
nitrite levels, was comparable among all treatments. Only the [N] group had
significantly lower nitrite levels during the final days of the experiment. But since the
[LA] group had a very high survival despite high nitrite levels, it can be suspected that
elevated nitrite concentrations were not the sole reason for low survival in the inert diet
46
groups. It may be possible that the shrimp fed with an additional live diet were better
capable to survive in water with lower quality.
The [N] and [LA] groups had a much higher survival (91 ± 7% and 94 ± 6%, respectively)
than Seychelles et al. (2018) reported for P. vannamei reared until PL6 and fed with
enriched nematodes Panagrolaimus sp. (NFS 24-5) and live Artemia (44 ± 24% and 33 ±
1%, respectively). Higher survival rates in later PL stages are expected, as larvae become
more resistant to stress after the critical PL6 stage when gills and excretory organs
become fully functional (McGraw et al., 2002).
As mentioned above, nematodes and Artemia can feed on bacteria from the biofloc
(Brüggemann, 2012; Luo et al., 2017; Yao et al., 2018) and could thereby bioencapsulate
valuable probiotics. Lakshmi et al. (2013) suggest probiotics in the diet of shrimp to
increase their health and resistance to stress and diseases. It is plausible that the live
diet groups had increased ingestion of probiotics from the biofloc, which could have
improved their survival in elevated nitrite levels compared to the inert diet groups.
Live diets play an important role in the feeding of early shrimp larvae, and complete
substitution of live Artemia has led to a reduction in survival and overall growth
performance in several studies (Hoseinifar & Zare, 2013; Sorgeloos et al., 1998). The
underlying issues and required nutrients of penaeid shrimp that undergo complicated
life cycles are complex and not yet fully understood (Robinson et al., 2005). However,
one identified problem that inert diets often pose, is the leaching of nutrients to the
water that can impair growth and survival but also degrades water quality (Kolkovski et
al., 2009; Villamar & Langdon, 1993). Gamboa-Delgado & Le Vay (2009) achieved good
results with a co-feeding regime of inert diets and live Artemia, in which nutritional
deficiencies of unenriched Artemia nauplii were compensated by specially formulated
diets rich in essential nutrients. Artemia are known to be very digestible and it has been
observed that penaeid larvae modulate their enzyme content in the gut in response to
dietary quality (Le Vay et al., 2001). Therefore, a live diet supplement may be beneficial
for the digestion of inert diets, resulting in better nutrient uptake and increased health
of the shrimp. The results of this experiment certainly support this hypothesis in regard
47
to health, as final survival in the live diets groups were near twice as high as in strictly
inert diet groups.
However, growth parameter results of wet weight, length, and RGR were statistically
significant higher in the inert diet groups. The PL from the [C] and [AA] groups were on
average 55.5% heavier at the end of the experiment. Although it must be noted, that the
PL were weighed in their wet state directly after the experiment, assuming that the
remaining water content was not perfectly identical among the groups. Therefore, wet
weight results should be interpreted with reservation. The length measurements on the
other hand stem from a very large sample size ([C]: n = 175; [AA]: n = 189; [N]: n = 222;
[LA]: n = 246) and are much more robust. The data shows the same tendency, that PL
from the inert diet groups are significantly different and on average 9.1% longer at the
end of the experiment. The RGR also revealed a significant difference between live and
inert diet groups, with the latter growing on average 2.5% more per day. But since the
RGR is also based on the wet weight measurements, this result must also be interpreted
with reserve. But the accumulated findings of the growth parameters point to the
conclusion that PL from the two inert diet groups [C] and [AA] were significantly larger
at the end of the feeding trial than PL that received a supplement of live diets.
The differences in size could be explained with the decreased PL density, due to the high
mortalities that occurred in the inert diet groups. In addition, the survivors received an
excessive amount of feed since the mortalities were unnoticed and the DF input was not
adjusted. Results by de Lorenzo et al. (2016) indicate longer larvae length at lower
stocking densities during the hatchery phase from mysis to PL5. Other studies with older
PL also verified that the growth of P. vannamei increases with decreasing densities
(Krummenauer et al., 2011; Moss & Moss, 2004).
There is also evidence that larger PL are more capable to survive adverse husbandry
conditions such as elevated nitrite levels (Jayasankar et al., 2009; Laramore et al., 2001;
Ramírez-Rochín et al., 2017). It is therefore plausible that the groups with the lowest
final survival had a larger proportion of heavier and longer individuals at the end of the
48
experiment. An involuntary selection of such animals would thereby skew the results of
the growth parameters towards the inert diet groups, which had the lowest survival.
Sorgeloos et al. (1998) reviewed the use of Artemia in the larval culture of several
crustaceans and concluded that they provide an adequate feed with high nutritional
value, especially when they are enriched with certain essential nutrients. More recent
studies emphasize the benefits of co-feeding regimes with inert diets and live Artemia
(Gamboa-Delgado & Le Vay, 2009; Zelaya et al., 2007). The results of this experiment
verify these early findings, as the survival of the PL fed with live Artemia were very high
compared with the inert diets and only the growth parameters were slightly reduced.
Seychelles et al. (2018, 2017) have already shown that the nematode species
Panagrolaimus sp. (NFS 24-5) is capable to replace Artemia nauplii during the early
larval stages of P. vannamei rearing. Nölting et al. (2017) successfully and completely
replaced Artemia for PL12-PL33 in a zero water and partial biofloc nursery. The results
considering the final survival in this study from PL10 - PL30 in complete biofloc system
confirm the recent findings. In a co-feeding regime of inert and live diets in a biofloc
system, nematodes can be used as a supplement replacing live Artemia nauplii. In
addition to their good performance to support shrimp survival, beneficial effects on the
water quality were also observed. The underlying mechanisms of this observation should
be further investigated, as they may prove to be another advantage of this nematode
species.
The simple preparation and application of desiccated nematodes outperforms the use of
Artemia cysts in terms of time and effort needed. Although it was not quantified in this
experiment, the preparation of live Artemia nauplii was the most time-consuming task
and the one that needed most labour. The procedure included the preparation of the
hatching tank, the hydration of cysts for 24 h, the harvest of nauplii, the separation of
nauplii from shells and the quantification of live nauplii. In comparison, the desired
quantity of nematodes only needed weighing and hydration in water for 30-60 min.
49
The results of the stress test revealed no differences among the groups in survival after
exposure to zero salinity. Since there was high mortality in the groups fed with inert
diets at the end of the feeding trial, it can be suspected that only the strongest survived
and were hence subjected to the salinity stress test. This involuntary pre-selection of
strong individuals would explain the lack of differences between the inert and live diet
groups. It is also possible that the duration of exposure (45 min) was not exerting
sufficient stress on the PL and that longer exposure times would have revealed
differences in survival. To evaluate the larval quality, salinity stress survival is an
important parameter that can ensure that PL will be resistant to transportation and
grow-out in a farm. Values of above 75% are required by most aquaculture enterprises
(FAO, 2003), and all treatments in this experiment had high survival values in the
salinity stress test that fulfil this criterion.
The body composition analysis returned similar results for lipids, ash, and protein in all
groups. The [LA] group had slightly higher ash levels. Unfortunately, not enough
material was available to analyze replicates and thus, no conclusion can be taken. In
further experiments, more emphasis should be given on such analysis approaches in
order to better understand the effects of live diets on body composition.
4.3. Final Conclusions
PL fed with a supplement of live Artemia or nematodes had significantly higher survival
but slightly lower growth than PL from control and artificial Artemia groups. The
nematode species Panagrolaimus sp. (NFS 24-5) can completely replace Artemia nauplii
in a co-feeding regime during the nursery phase in a zero-water exchange biofloc system.
In addition, beneficial effects on the biofloc and on the water quality were also observed
in the live diet groups. Further experiments investigating the complex interactions of
live diets, biofloc, water quality, and shrimp health should be designed to better
understand the underlying processes.
50
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Evaluation of Nematodes and Artificial

Artemia as Feed for Pacific White

Nils Phillip Sommer

A Master Thesis in Aquaculture and Fisheries

Faro, January 2019

Nils Phillip Sommer

Dissertation for the Attainment of the Master’s Degree in

Thesis was supervised by Dr. Cláudia Aragão

Faro, January 2019

Declaração de autoria de trabalho

Declaro ser o autor deste trabalho, que é original e inédito. Autores e

Universidade do Algarve, 30 Janeiro de 2019

© 2019 Nils Phillip Sommer

A Universidade do Algarve tem o direito, perpétuo e sem limites geográficos, de arquivar e

Keywords: Penaeus vannamei, nematodes, artificial Artemia, biofloc, nursery

Nas últimas décadas a produção aquícola global experimentou um imenso crescimento

Os produtos artificiais de Artemia já estão comercialmente disponíveis mas sua

A dieta de nematode também teve níveis de nitrito significativamente menores,

Palavras-chave: Penaeus vannamei, nematodes, Artemia artificial, biofloco, viveiro

AA Artificial Artemia m Meter

2. Material and Methods........................................................................................................................... 1

Table 1: Feeding Schedule. .................................................................................................. 20

1.1. Aquaculture Overview

One important production segment of aquaculture is the group of marine crustaceans,

1.2. Penaeus vannamei

1.3. History of Penaeus vannamei Aquaculture

1.4. Production Techniques of Penaeus vannamei

The aquaculture production of P. vannamei is fundamentally based on the grow-out of

1.5. Current Issues and Measures

The intensification of aquaculture brings with it an immense excess of organic

Due to the extremely fast growth of heterotrophic bacteria in carbon-rich conditions,

The heterotrophic bacterial reaction also consumes a modest amount of alkalinity as an

The natural diet of shrimp larvae generally consists of diverse phytoplankton,

Furthermore, large scale mass production methods of a specific nematode species,

2.1. Preparation of the Experimental Facility

Figure 2: A: Holding Tank; B: Bucket with meshed overflow.

Day 1- Day 5 (PL11-PL14) *

Day 6 – Day 9 (PL15 –PL18) **

Day 10 – Day 13 (PL19 - PL22) **

Day 14 – Day 21 (PL23 – PL30) **

Dextrose (g) - - - - - - - - - - - - - - 2.5 2.0 1.0 1.0 1.0 1.0

2.3. Feed Preparation

Figure 3: Diagram of Artemia hatching tank.

2.3.3. Preparation of dry feed and artificial Artemia

2.4. Water Quality Analysis

2.6. Final Sampling

RGR (%/day) = (eg−1) × 100, (1)

2.7. Statistical Analysis

3.1. Water Quality Parameters

3.1.2. Dissolved Oxygen

Wet Weight Total Length RGR

4.1. Water Quality Parameters

4.2. Larval Performance

4.3. Final Conclusions

Avnimelech, Y. (1999). Carbon / nitrogen ratio as a control element in aquaculture systems.

Bermudez-Almada, M. C., & Espinoza-Plascencia, A. (2012). The Use of Antibiotics in Shrimp

Briggs, M. (2006). FAO. Cultured Aquatic Species Information Programme. Penaeus

Browdy, C. L. (1998). Recent developments in penaeid broodstock and seed production

Brüggemann, J. (2012). Nematodes as Live Food in Larviculture - A Review. Journal of the