Comparison of Fatty acids profile of Marine species off Namibia

COMPARISON OF FATTY ACIDS PROFILE
OF BENGUELA SPECIES OFF NAMIBIA.
KANYIKI VILHO ROYAL
BACHELOR OF SCIENCE (HONOURS) IN FISHERIES AND AQUATIC SCI.
UNIVERSITY OF NAMIBIA
SUPERVISOR: DR. IITEMBU JOHANNES

COMPARISON OF FATTY ACID PROFILE OF BENGUELA SPIECIES
i
Declaration & AcknowledgementDeclaration & Acknowledgement
I hereby declare that this final year project report, submitted to University of Namibia as a
partial fulfillment of the requirements for the Bachelor of Science Honors degree in Fisheries
and Aquatic Sciences. I also certify that the work described here is entirely my own except
for summaries whose resources are appropriate cited in the references.”
Name: Vilho R. Kanyiki
I acknowledge and honor the Grace of God upon my life, thanks to my parents and the entire
Kanyiki family for your unconditional love. Special thanks to my supervisor Dr. Iitembu JA for
making this happen. With love, appreciations to my fellow classmates for encouragement and
support.

ii
AbstractAbstract
The Benguela ecosystem is important to the fishery off Namibia. Helicolenus dactylopterus,
Synagrops microlepis & Chlorophthalmus agassizi are important species in the Benguela
ecosystem. The three species are found in the diet of many predatory fish species in marine
waters off Namibia, live at an overlapping depth ranges and have common prey in their diets.
The objective of this study was to compare the fatty acid profiles of these three species.
Multivariate tests revealed significant differences in the storage of MUFA, PUFA and SFA
profile between the three species, displaying that fatty acids in fish differ between species.
All the species had higher PUFA compared to MUFA and SFA, agreeing that Polyunsaturated
Fatty Acids are the most active and dominant fatty acids in marine fish. This indicate that
although these species have prey in their difference, there significant differences is in their
dietary source.
Keywords: Fatty Acids, MUFA, PUFA, SFA, Helicolenus dactylopterus, Synagrops microlepis
& Chlorophthalmus agassizi.

iii
Table of contentsTable of contents
Abstract ii
1. Introduction 1
2. Material and Methods 3
2.1 Study area and field sampling 3
2.2 Laboratory analysis 4
2.3 Statistical analyses 4
3. Results 5
3.1 FA profile 5
3.2 MonoUnsaturated Fatty Acids 7
3.3 PolyUnsaturated Fatty Acids 7
3.4 Saturated Fatty Acids 7
4. Discussion 8
5. Reference 11
6. Appendices 15

1
The Benguela ecosystem which extend over three countries (South Africa, Namibia and
Angola) is one of the coastal upwelling systems in the world (Coetzee et al. 2008).
High phytoplankton productivity in the nutrient rich upwelling waters is the basis for the
highly productive Benguela ecosystem. The Benguela ecosystem is important to the fishery
communities off Namibia, it supports wide range of marine fishery with catches and production
of commercial species of over a million tons per year, (Sakko 1998). Studies conducted
in the Benguela ecosystem allow better understanding of liveliness of marine species and
contribute knowledge toward their diverse exploration, hence find strategic managements that
will sustain and protect their population. Jacopever (Helicolenus dactylopterus), Thinlip splitfin
(Synagrops microlepis) and Shortnose greeneye (Chlorophthalmus agassizi) fish are important
species in the Benguela ecosystem. They are studied to live at an overlapping depth ranging
from 100 to 1000m deep, (Anastasopoulou et al. 2005; Hamukuaya et al. 2001; Sequeira
et al. 2009). The Jacopever is a benthic deep-water fish which is widespread in the eastern
and north Atlantic Ocean, (Sequeira et al. 2009). Shortnose greeneye is a demersal species
that live in mud and clay bottoms and is very abundant in the Western Atlantic Ocean,
(Anastasopoulou et al. 2005). The Thinlip splitfin is a bathypelagic species normally found
in the Eastern Atlantic off the coasts of Walvis Bay, (Hamukuaya et al. 2001). These three
Benguela species have common prey in their diets, both benthic and pelagic organisms such
as crustaceans, fishes, cephalopods, and echinoderms, (Eschmeyer and Dempster 1990).
Chlorophthalmus agassizi however showed a mixed feeding strategy, exploiting a wide range of
prey including mesopelagic, benthic and endo-benthic organisms, (Anastasopoulou and Kapiris
2007). The three species are found in the diet of many predatory fish species (e.g. Hake
species) in marine waters off Namibia. Literatures indicates early exploration of these species,
for example Macpherson & Roel (1987) studied the daily ration and feeding periodicity of
some fishes off the coast of Namibia and trophic relationships in the demersal fish community
off Namibia. The findings of the above cited studies were based on stomach-content analyses.
Therefore, the use of Fatty acids analysis have improved our understanding of many marine
ecological relationships, they provide long-term and time-integrated dietary information about
consumers, (Koussoroplis et al. 2010). Fatty acids have previously been used to examine
qualitative aspects of food webs, energy transfers, and predator prey relationships, (Sara et al.
2004). Hence the method of exploring consumer diets based on fatty acid profiles represents
an additional and complementary approach to those already used and may shed further light
on the trophic
1. Introduction1. Introduction

dynamics of closely related species that inhabit the same oceanic regions, (Iitembu and
Richoux 2014). The difference of fat acids compositions of marine fish depends on diet,
geographic conditions, body length, sex, species, and fat content, (Baris et al. 2014).
Remarkably, fatty acids have been used as qualitative markers to trace or confirm predator-
prey relationships in the marine environment for more than thirty years, (Dalsgaard et al.
2003). Fatty acids are also known to play a number of key roles in metabolism (storage
and transport of energy), as essential components of all membranes, and as gene regulators
(Drevon 2010). As part of complex lipids, fatty acids can be saturated, monounsaturated
or polyunsaturated (Drevon 2010). Robin et al. (2003) studied that the fatty acid (FA)
content of fish reflect fatty acid composition of the diet. Hence the incorporation of FA into
tissues is modulated by various metabolic factors, and final composition will depend upon the
initial FA content, cumulative intake of dietary fatty acids, growth rate and duration, (Robin et
al. 2003). The objective of this study was to compare the fatty acid profiles of these three
species. The specific objectives of the study were to compare fatty acid profiles within and
between the species. This study therefore contributes towards research effort of understanding
their interspecific trophic relationships. Knowledge from this study is useful toward sustainable
fisheries practice especially in understanding prey-predatory species interactions.
2
1. Introduction1. Introduction
contributes towards research effort of
understanding their interspecific trophic
relationships..

Data used in this study were collected by the Ministry of Fisheries and Marine resources,
during demersal survey (2012). Sample were collected from pre-determined survey stations.
The collections were completed during a hake biomass survey (11 January–25 February
2011) on board MV Blue Sea I and a monkfish biomass survey (16–27 December 2011)
on board RV Welwitschia. Sampling for all fish was opportunistic, with the general aim of
obtaining a wide size distribution of each species. At each station, 1–10 individuals (depending
on availability) were selected, (see Iitembu and Richoux 2016).
2.1 Study area and Field Sampling
Figure 1. Sampling stations along the Atlantic Namibian coastline
3
2. Materials & Methods2. Materials & Methods

The laboratory analyses of the FA extract were done at Rhodes University. A small section of
white muscle was removed from the anterodorsal region of each frozen fish, then lyophilized
at −60 °C for 24 hour. The Lyophilized samples were first grounded individually with a mortar
and pestle into a fine powder then placed in a glass test tube, after which chloroform (with
butylated hydroxytoluene) was added, the tube were later flushed with nitrogen and then
stored at −20 °C. Total lipid were extracted in 8:4:3 (v/v/v) of CHCl3/methanol/water. A
neutral lipids of tissues were extracted from the total lipids using column chromatography on
silica gel, (See Iitembu and Richoux 2016).
MANOVA was used to determine whether there is significant differences in the Fatty acids
profiles of the three species. The Tukey HSD was used to determine the Fatty acids that
contributed more to the significant difference of the FA profiles between the three species.
2.2 Laboratory Analysis
2.3 Statistical Analysis
4
2. Materials & Methods2. Materials & Methods

A total of 24 samples of the three species collected along different station off the Benguela
region were analyzed for fatty acid (FA) profiles. Fifty three (53) FAs ranging from 14 to
24 carbon atoms in length, were identified from the samples. Analyses was limited to most
abundant 30 FAs (Table 1) that were detected in amounts >1 % in all species. All the
species had higher PolyUnsaturated Fatty Acids (PUFA) compared to MonoUnsaturated Fatty
Acids (MUFA) and Saturated Fatty Acids (SFA) (Fig 2). H. dactylopterus showed higher
PUFA and SFA content compared to other species while C. agassizi had a higher MUFA
content (Fig 2). A significant difference between the MUFA, PUFA and SFA profiles of the
three species was observed, (p<0.05), (p<0.05) and (p<0.05).
There was a significant differences between the FA profiles of the three species (MANOVA,
Wilk’s λ=33.22, p<0.05). A Tukey HSD of the FAs indicated that 14:0 was significantly
different between H. dactylopterus and S. microlepsis (p<0.05) and between C. agassizi
and H. dactylopterus (p<0.05). 18:1w7 was significantly different between C. agassizi and
H. dactylopterus (p<0.05) and between H. dactylopterus and S. microlepsis (p<0.05).
Tukey HSD indicated a significant difference in 22:6w3 of C. agassizi and H. dactylopterus
(p<0.05), and between H. dactylopterus and S. microlepsis (p<0.05).
3.1 Fatty Acids Profiles
5
3. Results3. Results
Figure 2. Average composition of MUFA, PUFA AND SFA, observed from the 30 FAs.

6
FAs Chlorophthalmus agassizi
(n=12). 12-20 cm
Helicolenus dactylopterus
(n=9). 12-14 cm
Synagrops microlepsis
(n=3). 13-15 cm
Mean(SD) Mean(SD) Mean(SD)
16:1w7
17:1w7
18:1w9
18:1w7
18:1w5
20:1w9
22:1w9
20:4w3
20:5w3
20:2w6
20:3w6
20:4w6
20:3w3
21:5w3
18:2w6
18:4w3
22:2
22:4w6
22:5w6
22:5w3
22:6w3
14:0
15:0
16:0
(i)17:0
17:0
18:0
20:0
(i)15:0
(ai)7:0
21:0
.48(1.17)
.38(.26)
15.07(10.37)
11.19(14.50)
.69(1.23)
3.27(2.93)
3.64(2.24
.55(.23)
9.01(2.51)
.20(.10)
.03(.06)
1.73(1.08)
.08(.08)
.00(.00)
.18(.36)
.40(.42)
.00(.00)
.17(.12)
.44(.29)
2.35(.94)
24.43(14.31)
5.13(2.44)
.314(.12)
15.29(11.49)
.29(.15)
.62(.17)
3.22(2.47)
.25(.12)
.12(.07)
.09(.06)
.08(.11)
.33(.65)
.39(.22)
7.58(6.23)
.79(1.44)
.059(.11)
1.92(1.92)
.64(.89
4.09(1.52)
.07(.13)
.09(.19)
.08(.23)
4.10(1.53)
.07(.13)
.17(.41)
.32(.50)
.00(.00)
8.24(2.75)
.48(.47)
1.24(.55)
3.29(1.43)
39.10(7.36)
1.25(.65)
.43(.18)
21.73(2.34)
.41(.34)
.63(.27)
5.90(2.25)
.29(.65)
.07(.06)
.16(.26)
.02(.06)
1.79(2.71)
.38(0.87)
25.91(15.07)
3.19(.24)
13.68(23.41)
4.70(4.14)
3.26(.37)
14.03(.06)
.56(.06)
.18(.02)
13.01(.02)
.63(.08)
9.04(.06)
5.53(.59)
.00(.00)
.00(.00)
.00(.00)
8.04(.06)
.17(.01)
1.62(.17)
8.24(.99)
7.83(.82)
.28(.02)
15.59(13.53)
.30(.05)
.48(.05)
4.14(3.59)
.37(.04)
.13(.01)
.09(.01)
.08(.09)
Table 1. The 30 FAs detect in amount above 1% used in the parametric analysis.

There was significant difference between the MUFA profiles of the three species (MANOVA,
Wilk’s λ= 0.129, p<0.05). A Tukey HSD indicated that 18:1w9 was significantly different
between H. dactylopterus and S. microlepsis, (p<0.05); 18:1w7 was significantly different
between C. agassizi and H. dactylopterus (p<0.05) and 22:1w9 was significantly different
between H. dactylopterus and C. agassizi, (p<0.05) and between H. dactylopterus and
S. microlepsis (p<0.05).
There was significant difference between the PUFA profiles of the three species (MANOVA,
Wilk’s λ= 0.044, p<0.05). A Tukey HSD indicated that 20:4w3, 20:5w6 and 22:2 were
significant different between C. agassizi and H. dactylopterus, (p<0.05), (p<0.05) and
(p<0.05); 22:6w3 was significantly different between S. microlepsis and H. dactylopterus,
(p<0.05) while Fatty acid 22:5w3 was significantly different between C. agassizi and S.
microlepsis, (p<0.05).
There was significant difference between SFA profiles of the three species (MANOVA,
Wilk’s λ= 0.101, p<0.05). A Tukey HSD indicated that Fatty acid 14:0 was significantly
different between the SFAs of C. agassizi and H. dactylopterus, (p<0.05) and between S.
microlepsis and H. dactylopterus (p<0.05) while 15:0 was significantly different between S.
microlepsis and C. agassizi (p<0.05).
3.2 MonoUnsaturated Fatty Acids
3.3 PolyUnsaturated Fatty Acids
3.4 Saturated Fatty Acids
7

This study aimed at comparing the fatty acid profiles of Helicolenus dactylopterus, Synagrops
microlepsis and Chlorophthamus agassizi. Multivariate tests revealed significant differences in
the storage of MUFA, PUFA and SFA profile between the three species. All the species had
higher PUFA compared to MUFA and SFA. H. dactylopterus showed higher PUFA and SFA
content compared to other species while C. agassizi had a higher MUFA content.
A mean comparison between the species FAs indicated a higher PUFA than MUFA and
SFA composition. The composition of the fatty acids in fish differ between species (Jalaludin
2013), depending on structure, properties, requirements and functions in the body (Baeza
2015). The results had reflected similarly with Baeza (2015) findings, the aquatic medium
is characterized by a wealth of PUFAs with fish containing always an elevated percentage of
PUFAs. The higher storage of PUFA in fish is general reflected by its demonstrated major
function in different aspects of the fish development, (Field 2003). They have been shown
to alter the expression of numerous genes involved in the metabolic function of the cell,
modulate the expression of a variety of genes coding for key regulatory proteins in metabolic
pathways such as those involved in digestion, glycolysis, glucose transport, inflammation, and
cellular communications, (Field 2003).
Palou (2007) highpoint Polyunsaturated Fatty Acids (PUFA) as most active and dominant
fatty acids in marine fish. This is acknowledged for their role as physiologically active factor
in many fish species to actively participate in gonad maturation, egg quality (Izquierdo et al.
2001) and larval growth of fish (Tulli and Tibaldi 1997).
Comparably, SFAs and MUFAs are heavily catabolized for energy in fish because they are
consumed in large amounts during growth, (Baeza 2015). They are identified in the body
of fish to be more structural in nature, and thus more rapidly influenced by changes in the
requirement of metabolic energy, (Koop- man et al. 1996, Iverson et al. 2002). Therefore,
the mobilization of SFA is required solely for provision of metabolic energy and less destined in
gonads and larval development of the fish, (Sargent et al. 1989). While MUFAs are crucial
stored to provide special conformational properties to the bio-membranes, and assist tissue
specific cells in reacting to external stimuli such as changing environmental temperatures and
light regimes (Sargent et al. 1993, Cook 1996).
8
4. Discussion4. Discussion

The results agrees with other studies on FAs, most marine fish lack delta -5 desaturase
activities needed to biosynthesize PUFAs and therefore have an absolute dietary requirement
for unsaturated FAs (Tocher and Ghioni 1999; Hastings et al. 2001; Nichols 2003).
Marine species present low enzymatic activity and depend almost completely on their diet to
obtain the main long-chain n-3 PUFAs, (Toucher 2010). Hence, unsaturated fatty acids may
be synthesized by animals but only to a limited extent and must be largely supplemented by
the diet (Steffens, 1997).
H. dactylopterus showed higher PUFA and SFA contents compared to other species. Although
very little information is available on the feeding habits of this species, studies indicated that
the species feeding strategy which, according to Macpherson (1985), is primary a daytime
predator feeding during a relative short period. Also a two clear dietary shifts (at 20 cm
and 28 cm TL) occur along H. dactylopterus life, (Sequeira et al. 2009). Nutritional needs
during the species growth changes, large individuals show major consumption of natantia which
are rich source of SFA, (Sequeira et al. 2009).
C. agassizi had a higher MUFA content compared to other species. The species is studied
to be an active carnivorous predator of benthic, an agile prey that exhibits a high feeding
activity (Anastasopoulou & Kapiris 2007). Stomach content analysis indicated that fish
and crustacean are abundant prey but a high abundance of plant detritus (plant sources of
fat tend to be very high in monounsaturated and polyunsaturated fats, (Assy et al. 2010))
found in their stomachs, confirmed a mixed type of diet, (Anastasopoulou & Kapiris 2007).
A significant differences was found between the MUFAs of the three species. Asclepic acid
(18:1w7) and Eurisic acid (22:1w9) were significantly different between H. dactylopterus
and C. agassizi. Asclepic and Eurisic acids belongs to the class of chemical entities known
as long-chain fatty acids, these are fatty acids with an aliphatic tail that contains between 13
and 21 carbon atoms (Lambertsen 1977). Studies (Assy et al. 2010) outlined that some
marine organisms lack the ability to introduce double bonds in long-chain fatty acids beyond
carbon 9 and 10, hence this might had considerable influenced the significant difference
between the different species.
There was significant difference between the PUFA profiles of the three species. DHA
(22:6w3) was significantly different between S. microlepsis and H. dactylopterus. Fatty
acid such as EPA and DHA that been found in marine fish are originally obtain from the
phytoplankton and also seaweed that include in their food chain (Cavington 2004).
9

Therefore, the difference in DHA could be a result of species different feeding activities as
studies indicates that the S. microlepsis migrate towards the surface at night for feeding
purpose (Heemstra 1984) which allow it to feed on the numerous floating populations of
plankton species which contain significant concentrations of EPA and DHA.
A significant difference was found between the SFA profiles of the three species. Myristic acid
(14:0) was significantly different between H. dactylopterus and S. microlepsis. Myristic acids
are found in all fish fats, and probably originate in marine phytoplanktonic algae (Lambertsen
1977). S. microlepsis storage of relatively high fat content (Iitembu and Richoux 2014)
could mean a high incorporation of 14:0 SFA hence reflect the difference in the storage
between the different species.
The findings of the study has shed light on the exploration of fish feeding based on fatty
acids (FAs). As observed, the species has more influence on the difference of fat acids
compositions between marine fish with additions to diet, geographic conditions, body length, sex
and fat contents. Studies have established that specific habits and characteristics from different
fish families, such as nocturnal/diurnal habits, anatomical and physiological characteristics
generally present differences in intestinal enzyme secretion metabolism, which influence nutrient
digestion and absorption processes, (Logato 1998). Thus, in addition to fish characters,
genetic potential and fatty acid enzymatic biosynthesis, can influence the final FA profile in
muscle tissue which can vary between species, (Toucher 2003).
More advanced and directed scientific studies must be conducted on the Benguela ecosystem
to document and cover all the important aspects of the environment interactions from fish to
planktons. Fish exploration is key to sustainable exploitation and truthful management practices
of marine environments. Hence, further studies should be developed were Stomach contents
analysis can be used together with fatty acids as quantitative and qualitative markers to trace
or confirm predator-prey relationships in the marine environment.
10

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1. Fifty three (53) FAs ranging from 14 to 24 carbon atoms in length, were identified
from the samples.
15
6. Appendixes6. Appendixes
Shorthand Common name Systematic name
14:0
14:1
i-15:0
ai-15:0
15:0
i-16:0
15:1
ai-16:0
16:0
16:1w7
16:1w5
i-17:0
ai-17:0
16:3w3
17:0
16:3w4
17:1w7
16:4w3
16:4w1
18:0
18:1w9
18:1w7
18:1w6
18:1w5
18:2w6
18:2w4
18:3w6
18:3w4
18:3w3
18:4w3
18:4w1
Myristic acid
Palmitic acid
Palmitoleic acid
Myristoleic acid
Hexadecatrienoic acid
Margaric acid
Stearic acid
Oleic acid
Vaccenic (Asclepic acid)
Linoleic acid
Gamma-linoleic acid
Alpha- linoleic acid
Stearidonic acid
Tetradecanoic acid
Pentadecanoic acid
Hexadecanoic acid
(Z) hexadec-9-enoic acid
c-9-tetradeconoic acid
7,10,13-hexacatrienoic acids
Heptadecanoic acid
4,7,10,13-hexadecatetraenoic acid
Octadecanoic acid
(Z)-octadec-9-enoic acid
11-octadecenoic
(9Z, 12Z)- octadeca-9,12-dienoic acid
(6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid
(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid
(6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid

16
Shorthand Common name Systematic name
20:0
20:1w9
20:1w7
20:2w6
20:3w6
20:4w6
21:0
20:3w3
20:4w3
20:5w3
22:0
22:1w11(13)
22:1w9
21:5w3
22:2
23:0
22:4w6
22:4w3
22:5w6
22:5w3
24:0
22:6w3
24:6w3
Arachidic acid
Gondoic acid
Dihomo-y-linoleic acid
Arachidonic acid
Eicosatrienoic acid
Eicosatetraenoic acid
Eicosapentaenoic acid
Behenic acid
Cetoleic acid
Erucic acid
Heneicosapentaenoic acid
Docosapentaenoic acid
Lignoceric acid
Docosahexanoic acid
Icosanoic acid
11-eicosenoic acid
(8Z,11Z,14Z)-icosa-8,11,14-trienoic acid
(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoic acid
11,14,17-eicosatrienoic acid
5,8,11,14,17-eicosatetraenoic acid
(5Z,8Z14Z,17Z)-icosa-5,8,11,14,17-pentanoic acid
Docosanoic acid
11-docosenoic acid
13-docosenoic acid
6,9,12,15,18-heneicosapentaenoic acid
(7Z)-docosa-7,10,13,16,19-pentaenoic aci
Tetracosanoic acid
(4Z)-docosa-4,7,10,13,16,19-hexaenoic acid
Station No. Latitude Longitude Depth
3
10
13
15
24
-23,3433
-24,3605
-24,9337
-25,805
-27,1898
13,48333
13,93067
13,96217
14,20383
14,67333
228
244
175
229
274
2. Sampling Stations along the Namibian Atlantic ocean

Stations Species Length
3
4
4
10
10
13
13
38
4
15
15
24
25
25
28
37
44
45
45
15
28
40
40
40
Chlorophthamus agassizi
13
1=¬3
13
15
15
15
15
15
13
12
13
18
19
20
18
14
11
15
13
13
17
12
13
14
Station No. Latitude Longitude Depth
25
28
37
38
40
44
45
-27,31
-28,115
-28,4762
-27,5878
-26,8402
-25,873
-25,8413
14,77833
14,86333
14,34133
14,41333
14,38717
13,766
13,55183
287
197
555
411
340
393
704
3. Species sampling stations and Lengths
17

Shortnose greeneye (Chlorophthalmus agassizi)
Thinlip splitfin (Synagrops microlepsis)
Jacopever (Helicolenus dactylopterus)
4. Species Images
18

19
7. Author details7. Author details
Vilho Royal Kanyiki
Hons degree in Fisheries and Aquatic science.
Faculty of Agriculture and natural resources.
University of Namibia
email: royalkanyiki995@gmail.com
Tel: +26481 3934633

Comparison of Fatty acids profile of Marine species off Namibia

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