Differences in composition and fatty

acid contents of different rainbow trout

( Oncorhynchus mykiss ) strains in
similar and contrasting rearing
conditions Michail I. Gladyshev
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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Differences in composition and fatty acid contents of different rainbow

trout (Oncorhynchus mykiss) strains in similar and contrasting
rearing conditions
Michail I. Gladyshev a, b, *, Alexander A. Makhrov c, Nadezhda N. Sushchik a, b,
Olesia N. Makhutova a, b, Anastasia E. Rudchenko b, Dmitrii A. Balashov d,
Evgenii V. Vinogradov d, Valentina S. Artamonova c
a
Institute of Biophysics of Siberian Branch of Federal Research Center “Krasnoyarsk Science Center” of Russian Academy of Sciences, Akademgorodok, 50/50,
Krasnoyarsk 660036, Russia
b
Siberian Federal University, Svobodny av. 79, Krasnoyarsk 660041, Russia
c
A. N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninsky prospect, 33, Moscow 119071, Russia
d
Branch for the Freshwater Fisheries of VNIRO (VNIIPRKh), Rybnoe 141821, Russia

A R T I C L E I N F O A B S T R A C T

Keywords: Fatty acid (FA) composition and contents of fillets of seven strains of rainbow trout Oncorhynchus mykiss, reared
Eicosapentaenoic acid in the same farm and sampled at the same time were studied. Three strains were compared with those from
Docosahexaenoic acid another farm as well as with outbreed and wild fish of the same fish species. In general, FA composition of farmed
Marker fatty acids
fish reflected their diet composition. Nevertheless, a genetics factor (belonging to a certain strain), also was
Nutritive value
found to contribute to fish FA composition. Rainbow trout of strain Steelhead appeared to have comparatively
higher ability to regulate their FA composition, in particular, to retain high contents of physiologically important
eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids in biomass despite variations of
their diet contents. Differences in contents of the EPA + DHA of different strains, found in this study, likely
provided evidence for a potential of selective breeding of rainbow trout strains with a high EPA and DHA, which
are of key importance for humans’ diet. Besides, FA markers, which allow differentiating wild and farmed
rainbow trout, were revealed.

1. Introduction However, in last decades, a tendency of decreasing LC-PUFA content

Long-chain polyunsaturated fatty acids of omega-3 family (LC-
PUFA), namely eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic
(22:6n-3, DHA) acids have been recognized as essential components of
human diet, which provide numerous health benefits, including pre­
vention of cardiovascular diseases (Calder, 2018; Bernasconi et al.,
2021). The principal dietary source of EPA and DHA for humans is fish
(Robert, 2006; Tacon and Metian, 2013; Gladyshev et al., 2013, 2015;
Tocher et al., 2019). Fish, reared in aquaculture, such as rainbow trout
Oncorhynchus mykiss have very high contents of EPA and DHA in their
edible parts – muscle tissue (filets), which determines its high nutritive
value for humans (Saglik Aslan et al., 2007; Stone et al., 2011; Turchini
et al., 2011, 2018).
in aquaculture of salmonids, including rainbow trout has occurred
because of replacing deficient fish oil, obtained from finite wild catch by
vegetable oils (Naylor et al., 2009; Turchini et al., 2009; Sprague et al.,
2016; Barry and Trushenski, 2020; Qian et al., 2020; Quiñones et al.,
2021).
One of the reasonable ways to support the high nutritive value of fish
species, reared in aquaculture, is a selection of strains that can have the
high contents of LC-PUFA in their biomass in spite of the increased levels
of plant oils in the feed (Naylor et al., 2009; Bell et al., 2010; Leaver
et al., 2011; Turchini et al., 2011; Shepherd and Jackson, 2013; Henri­
ques et al., 2014; Gladyshev, 2021; Gladyshev et al., 2022). However,
this selection could be successful only in a case of a high heritability of
LC-PUFA contents in muscle tissue.

* Corresponding author at: Institute of Biophysics of Siberian Branch of Federal Research Center “Krasnoyarsk Science Center” of Russian Academy of Sciences,
Akademgorodok, 50/50, Krasnoyarsk 660036, Russia.
E-mail address: glad@ibp.ru (M.I. Gladyshev).

https://doi.org/10.1016/j.aquaculture.2022.738265
Received 16 February 2022; Received in revised form 9 April 2022; Accepted 13 April 2022
Available online 19 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
M.I. Gladyshev et al. Aquaculture 556 (2022) 738265

In literature, there are data on differences in composition of LC-PUFA
between anadromous and landlocked Atlantic salmon (Salmo salar L.),
reared in similar conditions (Peng et al., 2003; Rollin et al., 2003;
Betancor et al., 2016). Indeed, n-3 LC-PUFA contents in muscle tissue of
this species is a highly heritable trait, h2 = 0.77 ± 0.14 (Leaver et al.,
2011; Horn et al., 2018), and there are differences in this trait between
different family groups of Atlantic salmon (Bell et al., 2010). Thus, there
is a potential of selective breeding of this species to increase LC-PUFA
contents in its muscle tissue (Horn et al., 2018).
In contrast, there is no data on the heritability of LC-PUFA contents
in muscle tissue of another important aquaculture species, O. mykiss, as
well as data on differences of LC-PUFA contents between strains of this
species. Thus, the aim of our study was a comparison of LC-PUFA con­
tents in muscle tissue of different strains of O. mykiss. We compared
different strains, reared both in similar and different conditions in three
fish farms from contrasting regions of Russia. Besides, LC-PUFA content
of a wild population of O. mykiss was measured for comparison.
proximate composition of the diets, reported by producers, are given in
Table 1.
Besides, five specimens of wild O. mykiss of 80–740 g (average mass
258 g) were caught in the Bystraia River (western part of Kamchatka
Peninsula). Species identity of these fish was confirmed on the basis of
sequencing of mitochondrial gene COI (Kirillova et al., 2021).
Fish were caught, euthanized using tricaine methane-sulfonate (MS-
222), 500 mg L− 1 (Topic Popovic et al., 2012), and sampled in accor­
dance with Federal Rules, including State Standard of Russian Federa­
tion, 33215-2014, 2016-07-01, and the protocol approved by the
Institutional Animal Ethical Committee on Biomedical Ethics of Siberian
Federal University (Russia).
For biochemical analyses, slices of fish white muscles of approxi­
mately 1.5–2 g, 2–3 cm below the dorsal fin were cut from an each
specimen. The muscle samples were immediately placed into a volume
of 3 mL of chloroform/methanol (2:1, by vol.) and kept until laboratory
analysis at − 20 ◦ C.

2. Materials and methods 2.2. Fatty acid analyses

2.1. Sampling Fatty acid analyses are given elsewhere (Gladyshev et al., 2020).
Briefly, lipids were extracted simultaneously with mechanical homog­
The study was performed in three reputable Russian fish farms, enization with chloroform/methanol mixture (2:1, v/v) three times. The
contrasting in rearing conditions. Farm 1 was situated in the zone of the dried lipids were hydrolysed under reflux at 90 ◦ C in a portion of
northern taiga; fish were reared in cages, placed in a large oligotrophic methanolic sodium hydroxide solution (8 mg/mL). Then the mixture
lake. Samples from Farm 1 were taken in November 31, 2018 and in May was added with an excess methanolic solution of 3% sulphuric acid and
19, 2019. Farm 2 was situated in the subtropical zone; fish were reared refluxed at 90 ◦ C for 10 min to obtain fatty acid methyl esters (FAMEs).
in concrete pools where water was pumped from artesian wells. Farm 2 The mixture was washed twice with portions of NaCl saturated solution,
provides fry planting material for many trout farms in Russia, including and FAMEs were extracted with a portion of hexane. FAMEs were
Farm 1. All samples from Farm 2 were taken in January 31, 2019. Farm 3 analyzed with a gas chromatograph equipped with a mass spectrometer
was situated in mountains; fish were reared in concrete pools where detector (model 7000 QQQ, or model 6890/5975C Agilent Technolo­
water was pumped from a river. Fish from Farm 3 were outbreed. gies, USA) and 30-m long, 0.25-mm internal diameter capillary HP-FFAP
Samples from Farm 3 were taken in November 03, 2019. columns installed in the both instruments. Detailed descriptions of the
In the farms, specimens of rainbow trout O. mykiss of average mass instrumental conditions are given in (Gladyshev et al., 2020). Data were
261 ± 22 g were sampled, which belonged to seven strains, common in collected and analyzed using MassHunter or Chemstation Software
Russian farms: Adler, Adler Amber, Steelhead, Kamloops, Augustin, (Agilent Technologies, USA). Peaks of FAMEs were identified by their
Donaldson and late spawning Steelhead. A history of breeding of these mass spectra, comparing them to those in the integrated database NIST
strains was reviewed by Artamonova et al. (2016). In general, 82 sam­ 2008 MS LIB (Revision Jan2010) and to those in the standard 37-FAMEs
ples of farm-raised fish were collected in three farms. Species identity of mixture (U-47885, Supelco, USA). The mixture was also allowed to es­
these fish were confirmed using microsatellite methods (Artamonova timate response factors for various FAMEs on the two instruments.
et al., 2016). In Farm 1, Farm 2 and Farm 3, commercial feeds, delivered FAMEs were quantified according to a peak area of the internal stan­
by three reputable international producers, were taken, designated dard, 19:0-FAME (Sigma-Aldrich, USA), which was added to samples
below as Diet 1, Diet 2 and Diet 3, respectively. Ingredients and prior to the lipid extraction, after addition of the first portion of chlo­
roform/methanol mixture.
Table 1
Ingredients and proximate composition of three commercial diets fed to rainbow 2.3. Statistical analysis
trout Oncorhynchus mykiss.
Diet 1 Diet 2 Diet 3 Standard errors (SE), Kolmogorov-Smirnov one-sample test for
normality DK-S, ANOVA with Tukey HSD post hoc test, Kruskal-Wallis
Ingredients Fishmeal, soy Fishmeal, soy Fishmeal, soy
protein meal, pea protein meal, protein meal,
test, Pearson’s correlation coefficient and multivariate discriminant
protein meal, wheat gluten meal, wheat gluten meal, analysis (MDA) (Legendre and Legendre, 1998) were calculated
wheat gluten fish oil, rapeseed fish oil, rapeseed conventionally, using STATISTICA software, version 9.0 (StatSoft, Inc.,
meal, fish oil, oil, vitamins, oil, vitamins, USA).
poultry fat, minerals minerals
rapeseed oil,
linseed oil, 3. Results
vitamins, minerals
Fatty acid composition of diets, i.e., mean values of levels of prom­
Proximate inent FAs (percent of total, ≥ 0.1%) are given in Table 2. Diet 1 had the
composition highest mean levels of 16:1n-7, 18:0, 18:2n-6 and 18:3n-3, but the
∑
Crude protein 42 41 44 lowest levels of 15-17BFA (sum of branched fatty acids with 15 and
(%) ∑
17‑carbon atom chains), 16PUFA (sum of polyunsaturated fatty acids
Crude lipid (%) 28 31 30 ∑
Ash (%) 4.7 5.0 5.3 with 16 carbon atoms), 18:1n-7, 18:4n-3, 20:1, 20:5n-3 and 22:5n-3
Carbohydrates 10 12 13 (Table 2). Diet 1 also tended to have a comparatively lower level of
∑
(%) 22:1 (Table 2). Diet 2 had the highest levels of 15:0, 17:0, 20:5n-3,
Gross energy 21.6 24.5 25.0 22:5n-6 and especially 22:6n-3, but the lowest levels of 16:1n-7, 18:0,
(MJ kg− 1) ∑
18:1n-9 and 18:3n-3 (Table 2). Diet 3 had the highest levels of 15-

2
M.I. Gladyshev et al. Aquaculture 556 (2022) 738265

Table 2
Mean values of percentages (% of total fatty acids ± standard error) and total contents (Total FA, mg g− 1 of dry weight) of fatty acids and sum of eicosapentaenoic and
docosahexaenoic acids (EPA + DHA, mg g− 1 of dry weight) in three diets, used in the three trout farms (n – number of samples). Normally distributed variables are
compared by ANOVA and Tukey HSD post hoc test; the other variables marked with* are compared by Kruskal-Wallis test. Means labelled with the same letter are not
significantly different at P < 0.05 according to the relevant test. When ANOVA or Kruskal-Wallis test are insignificant, letter labels are absent.
Fatty acid Diet 1 (n = 6) Diet 2 (n = 5) Diet 3 (n = 5)

14:0 1.5 ± 0.3 2.2 ± 0.2 2.0 ± 0.1

15:0* 0.1 ± 0.0A 0.5 ± 0.0B 0.2 ± 0.0A
16:0 16.9 ± 0.1A 17.0 ± 1.0A 12.7 ± 0.2B
16:1n-7 3.8 ± 0.1A 2.0 ± 0.1B 2.6 ± 0.1C
∑
15-17BFA 0.1 ± 0.0A 0.2 ± 0.0B 0.3 ± 0.0C
∑
16PUFA 0.4 ± 0.0A 0.8 ± 0.1B 1.0 ± 0.0C
17:0* 0.2 ± 0.0A 0.4 ± 0.0B 0.2 ± 0.0A
18:0 5.2 ± 0.1A 2.8 ± 0.1B 4.3 ± 0.2C
18:1n-9 35.1 ± 0.1A 32.4 ± 0.8B 43.6 ± 0.4C
18:1n-7 2.3 ± 0.0A 2.5 ± 0.0B 2.8 ± 0.0C
18:2n-6 17.4 ± 0.2A 14.5 ± 0.6B 14.1 ± 0.2B
18:3n-3 8.2 ± 0.2A 3.5 ± 0.3B 5.6 ± 0.3C
18:4n-3 0.2 ± 0.1A 0.4 ± 0.0B 0.6 ± 0.0B
∑
20:1 0.9 ± 0.1A 1.1 ± 0.1B 1.3 ± 0.0C
20:4n-6 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0
20:5n-3 2.0 ± 0.1A 3.7 ± 0.1B 2.9 ± 0.1C
∑
22:1 0.4 ± 0.1A 0.5 ± 0.0AB 0.6 ± 0.0B
22:5n-6* 0.0 ± 0.0A 2.1 ± 0.1B 0.0 ± 0.0A
22:5n-3 0.2 ± 0.0A 0.4 ± 0.0B 0.5 ± 0.0B
22:6n-3* 1.6 ± 0.2A 10.7 ± 0.4B 2.1 ± 0.1A
Total FA 209.3 ± 8.5A 78.5 ± 3.6B 191.5 ± 12.1A
EPA + DHA 7.4 ± 0.2A 11.2 ± 0.5B 9.6 ± 0.7B

∑ ∑
17BFA, 16PUFA, 18:1n-9, 18:1n-7 and 20:1, but the lowest level of from different farms (Fig. 1; Table 3). Root 1 discriminated best fish of
16:0 (Table 2). Diet 2 had the lowest total content (mg∙g− 1 of dry Adler and Adler Amber strains of Farm 1 from wild fish and fish of
weight) of fatty acids (Table 1). Diet 1 had the lowest contents (mg∙g− 1)
of EPA + DHA (Table 2).
The multivariate discriminant analysis (MDA), demonstrated sig­ Table 3
nificant differences in the FA compositions of wild and farmed trout Results of multivariate discriminant analysis of the fatty acid composition (% of
total FAs) of muscle tissue of wild and farmed rainbow trout of different strains.
Root 1 Root 2
15
Canonical R 0.996 0.990
Chi-square 1325 979
Degree of freedom 187 160
10
p <0.0001 0.0001

5 Means of canonical variables:

Farm 1, Adler 13.87 − 1.95
Farm 1, Adler Amber 14.07 − 3.14
Farm 1, Steelhead 12.16 − 2.93
0 Farm 2, Augustin − 1.39 11.90
Root 2

Farm 2, Donaldson − 10.70 − 1.32

Farm 2, late spawning Steelhead − 11.72 − 0.64
-5 Farm 2, Kamloops − 1.69 9.83
Farm 2, Adler − 2.77 6.05
Farm 2, Adler Amber − 6.37 − 0.44
Farm 2, Steelhead − 11.83 0.25
-10 Farm 3, outbred 3.51 − 1.10
Wild − 14.65 − 17.52

-15
Factor structure coefficients:
14:0 − 0.04 − 0.11
16:0 − 0.04 − 0.12
-20 16:1n-7 − 0.01 − 0.22
∑
-15 -10 -5 0 5 10 15 16PUFA − 0.02 − 0.10
Root 1 17:0 − 0.16 − 0.30
18:1n-9 0.21 0.20
18:1n-7 − 0.13 0.11
Fig. 1. Scatterplot of canonical scores for the two discriminant functions, Root
18:2n-6 0.31 0.22
1 and Root 2, after multivariate discriminant analysis (MDA) of the fatty acid 18:3n-6 0.16 0.11
composition (% of total FA): open circles – Farm 1, strain Adler, open squares – ∑
20:1 − 0.01 0.09
Farm 1, strain Adler Amber; open triangles – Farm 1, strain Steelhead; filled 20:2n-6 0.07 0.13
circles – Farm 2, strain Adler; filled squares – Farm 2, strain Adler Amber; filled 20:3n-6 0.12 0.25
triangles – Farm 2, strain Steelhead; filled diamonds – Farm 2, strain Kamloops; 20:4n-6 − 0.01 − 0.11
filled star – Farm 2, strain Augustin; open star – Farm 2, strain Donaldson; cross 20:5n-3 − 0.21 − 0.22
∑
– Farm 2, strain late spawning Steelhead; open diamonds – Farm 3, outbreed; 22:1 0.01 0.06
22:5n-6 0.02 − 0.10
oblique cross – wild fish. (For interpretation of the references to colour in this
22:6n-3 − 0.17 − 0.05
figure legend, the reader is referred to the web version of this article.)

3
M.I. Gladyshev et al. Aquaculture 556 (2022) 738265

Steelhead and late spawning Steelhead strains of Farm 2 (Fig. 1; Table 4

Table 3). Variables that gave the highest contribution to the first Results of two-way ANOVA comparing levels (% of total FAs) and contents (mg
discriminant function (Root 1) were 18:2n-6 and 18:1n-9, on the one g− 1 wet weight) of fatty acids, selected on the basis of the multivariate
hand, and 20:5n-3 and 22:6n-3, on the other (Table 3). Root 2 discriminant analysis (see text, Fig. 1 and Table 2 for details), which provide
discriminated best fish of Augustin and Kamloops strains of Farm 2 from most differences between rainbow trout of three strains, Adler, Adler Amber and
Steelhead, reared in two farms, Farm 1 and Farm 2: d.f. – degree of freedom, MS
wild fish (Fig. 1; Table 3). These differences in Root 2 were primarily due
– mean square effect for independent variables, F – Fisher’s test, P – significance.
to the contributions of 20:3n-6 and 18:2n-6 vs. 17:0, 16:1n-7 and 20:5n-
3 (Table 3). Variable Source of variation d. MS F P
f.
Average contents of EPA + DHA varied from 1.31 ± 0.20 mg g− 1 in
wild fish to 2.35 ± 0.09 mg g− 1 in fish from Farm 1, strain Adler Amber 18:2n-6, % Farm 1 211.428 201.26 0.00000
Strain 2 23.586 22.45 0.00000
(Fig. 2). All fish from Farm 1, as well as fish of strain Steelhead and strain
Farm × Strain 2 10.447 9.94 0.00030
late spawning Steelhead from Farm 2, and outbred fish from Farm 3 had interaction
significantly higher contents of EPA + DHA than the wild trout (Fig. 2). 20:5n-3, % Farm 1 51.539 104.33 0.00000
Fish of strain Adler Amber from Farm 1 had significantly higher contents Strain 2 31.185 63.13 0.00000
of EPA + DHA than fish of strains Adler, Augustin and Kamloops from Farm × Strain 2 7.593 15.37 0.00001
interaction
Farm 2 (Fig. 2). Average content of EPA + DHA for all farmed trout was
22:6n-3, % Farm 1 914.425 105.66 0.00000
2.03 ± 0.05 mg g− 1. Strain 2 272.679 31.51 0.00000
Three strains, Adler, Adler Amber and Steelhead, that were reared Farm × Strain 2 79.852 9.23 0.00049
both in Farm 1 and Farm 2, were compared using two-way ANOVA interaction
Total FA, mg Farm 1 1663.464 81.90 0.00000
regarding a number of FAs, revealed by MDA as principal contributors to
g− 1
the differences between farms and strains. In most cases, effects of the Strain 2 308.340 15.181 0.00001
farms, the strains and their combinations on FA levels (%) and contents Farm × Strain 2 251.815 12.398 0.00006
(mg g− 1), were statistically significant, except effects of farm × strain interaction
combinations on EPA and DHA contents, and the effect of strains on 20:5n-3, mg Farm 1 0.105 11.31 0.00168
g− 1
DHA content (Table 4). Means, related to the significant effects, are
Strain 2 0.053 5.76 0.00624
given in Fig. 3. Fish, reared in Farm 1, had significantly higher levels of Farm × Strain 2 0.006 0.706 0.50163
linoleic acid (18:2n-6, LA) than fish from Farm 2 (Fig. 3). Fish of strain interaction
Steelhead had significantly lower level of LA than fish of two other 22:6n-3, mg Farm 1 0.400 5.23 0.02745
g− 1
strains (Fig. 3). The highest levels of LA were characteristic of fish of
Strain 2 0.147 1.92 0.15955
strains Adler and Adler Amber from Farm 1, while the lowest levels of Farm × Strain 2 0.050 0.66 0.52400
this FA were characteristic of fish of strains Adler Amber and Steelhead interaction
from Farm 2 (Fig. 3). In contrast, mean levels of EPA and DHA of fish,
reared in Farm 1, were significantly lower, than those of fish from Farm
2 (Fig. 3). In turn, fish of strain Steelhead had significantly higher level wild trout are depicted in Fig. 4. Besides of the significant mean dif­
of EPA and DHA, than fish of two other strains (Fig. 3). Fish from Farm 1 ferences, minimum levels of 18:1n-9 and 18:2n-6 in the farmed fish were
had a significantly higher content of total FA (mg g− 1) as well as those of higher, than their maximum levels in the wild fish (Fig. 4). In turn,
EPA and DHA (Fig. 3). Fish of strain Steelhead had the lowest content of minimum levels of 18:0 and 20:4n-3 in the wild fish were higher, than
total FA, but fish of strain Adler had the lowest content of EPA (Fig. 3). their maximum levels in the farmed fish (Fig. 4). Levels of 20:5n-3,
The highest content of total FA was characteristic of fish of strain Adler, 20:2n-6 and 20:3n-6 in the farmed and wild fish overlapped only
reared in Farm 1 (Fig. 3). marginally (Fig. 4).
Fatty acids, with significantly different mean levels in the farmed and Seven strains of rainbow trout that all were reared in Farm 2 mark­
edly varied in a season of spawning, which is genetically determined for
each strain. To quantify the season of spawning, we used number of days
3 from the beginning of year to the peak of spawning. Fish of strain
a ab ad Augustin had the earliest season of spawning, started in August and
ab ab ab peaked in September. The latest season of spawning, in March, is
EPA+DHA, mg g-1

abc abc
2 characteristic of fish of strain late spawning Steelhead. Thus, fish of
bcd
bc bc strain Augustin had the shortest period, 250 days, and fish of strain late
c
spawning Steelhead had the longest spawning, 455 days. The fish strains
also varied in their average EPA + DHA content, from 1.6 to 2.1 mg g− 1
1 (Fig. 5). To consider likely relation between the season of spawning and
PUFA content in fish muscles, we calculated Pearson’s correlation co­
efficient between these two parameters: r = 0.91, p < 0.01, d.f. = 5.
Thus, fish strains with later season of spawning tended to accumulate
0
F1A F1M F1S F2A F2M F2S F2U F2K F2D F2V F3o W more essential PUFA in their muscle tissues, when rearing in the iden­
tical conditions.
Fig. 2. Average contents of sum of eicosapentaenoic and docosahexaenoic fatty
acids (EPA + DHA) in rainbow trout of different strains and farms, and in wild 4. Discussion
rainbow trout: F1A – Farm 1, strain Adler; F1M – Farm 1, strain Adler Amber;
F1S – Farm 1, strain Steelhead; F2A – Farm 2, strain Adler; F2M – Farm 2, strain In the studied farms, diets used for rainbow trout rearing, differed
Adler Amber; F2S – Farm 2, strain Steelhead; F2U – Farm 2, strain Augustin; significantly in their constituents. Diet 1, used in Farm 1, evidently
F2K – Farm 2, strain Kamloops; F2D – Farm 2, strain Donaldson; F2V – Farm 2, included comparatively higher proportion of vegetable oils, since it had
strain late spawning Steelhead; F3o – Farm 3, outbred family; W – wild trout. significantly higher percentage of 18:2n-6, LA (Naylor et al., 2009;
Means labelled with the same letter are not significantly different at P < 0.05
Hixson et al., 2014; Chaguri et al., 2017). In turn, Diet 2, used in Farm 2,
after ANOVA and Tukey HSD post hoc test. (For interpretation of the references
had comparatively higher proportion of fish oil, which was indicated by
to colour in this figure legend, the reader is referred to the web version of
this article.) significantly higher levels of 20:5n-3 (EPA) and 22:6n-3 (DHA) (Naylor

4
M.I. Gladyshev et al.
et al., 2009; Chaguri et al., 2017). Besides, Diets 1 and 3 had signifi­
cantly higher content of total FA (mg g− 1). Nevertheless, in spite of the
significantly lower content of total FA, Diet 2 had significantly higher
content of EPA + DHA (mg g− 1), which was evidently due to the higher
proportions of these acids in total FA.
The peculiarities of FA composition and contents of diets in general
5
Aquaculture 556 (2022) 738265
Fig. 3. Average levels (% of total FAs) and contents
(mg g− 1 wet weight) of fatty acids, selected on the
basis of the multivariate discriminant analysis (see
text, Fig. 1 and Table 2 for details), which provide
most differences between rainbow trout of three
strains, Adler, Adler Amber (designated as Amber)
and Steelhead (designated as Steel), reared in two
farms, Farm 1 and Farm 2. Means labelled with the
same letter are not significantly different at P <
0.05 after two-way ANOVA and Tukey HSD post
hoc test. F1 and F2 – Farm 1 and 2, respectively, A,
M and S – strains Adler, Adler Amber and Steel­
head, respectively. (For interpretation of the refer­
ences to colour in this figure legend, the reader is
referred to the web version of this article.)
were reflected in FA composition of reared fish. Indeed, according to the
multivariate discriminant analysis, FA composition of trout, reared in
Farm 1 differed from that of trout from Farm 2 primarily due to the
differences in levels of 18:2n-6, on the one hand, and 20:5n-3 and 22:6n-
3, on the other hand, while fish from Farm 3 had an intermediate po­
sition in the multidimensional space of FA. Moreover, mean levels of the
M.I. Gladyshev et al. Aquaculture 556 (2022) 738265

40
Median
25-75%
Min-Max
30

Major FA, %
20

10

0
16:0 16:1n-7 18:0 18:1n-9 18:2n-6 20:5n-3 22:6n-3

3
Minor FA, %

0
17:0 18:3n-6 18:4n-3 ∑20:1 20:2n-6 20:3n-6 20:4n-6 20:4n-3 ∑22:1 22:5n-6

Fig. 4. Mean levels of fatty acids (% of total FA) in farmed (open rectangles, number of cases, n = 82) and wild (grey rectangles, n = 5) rainbow trout, which differed
significantly (P < 0.05) according to Student’s t-test. Note different scales for major and minor FA.

was in general confirmed in our study, like in some other studies of

Fig. 5. Average contents of sum of eicosapentaenoic and docosahexaenoic fatty
acids (EPA + DHA, bars and left axis, m ± SE) in rainbow trout of different
strains from Farm 2 versus number of days from the start of year to the peak of
spawning (a proxy of spawning season, diamonds and right axis).
common strains, Adler and Adler Amber, reared in Farm 1 and Farm 2,
had the same significant differences, as those of diets, i.e., fish from
Farm 1 had in their biomass significantly higher levels of LA, originated
from vegetable oils, and significantly lower levels of EPA and DHA,
originated from fish oil.
However, although the well-known principle “you are what you eat”
rainbow trout (Barry and Trushenski, 2020; Quiñones et al., 2021), the
other factor, namely genetics (belonging to a strain), also was found to
contribute to fish FA composition. Indeed, FA compositions of fish of two
strains, Adler and Adler Amber, strictly reflected food composition, i.e.,
fish of these strains in Farm 1 (Diet 1) had significantly lower levels of
EPA and DHA than fish of these strains in Farm 2 (Diet 2). In contrast,
fish of Steelhead strain in Farm 1 and in Farm 2 had similar levels of
these physiologically important acids in spite of their different levels in
diets. Thus, fish of strain Steelhead likely had comparatively higher
ability to regulate their FA composition regarding levels of the essential
LC-PUFA. Thus, in this case an interaction of genotype and environment
took place.
The ability of some fish species, including rainbow trout to maintain
an optimal physiological level of LC-PUFA in muscle tissue under their
dietary deficiency via selective incorporation and/or synthesis from
18:3n-3 has been described by many authors (Sushchik et al., 2006;
Turchini et al., 2009; Leaver et al., 2011; Strandberg et al., 2015; Tocher,
2015; Chen et al., 2017; Gladyshev et al., 2018; Barry and Trushenski,
2020).
The studied farmed and wild trout had different nutritive values
regarding content of EPA and DHA (mg g− 1 of wet weight). Contrary to a
widespread opinion of consumers about a higher nutritive value of wild
fish compared to that of farmed species, in our study wild rainbow trout
had significantly lower EPA + DHA contents than majority of farmed
trout strains. However, in other studies of wild rainbow trout O. mykiss
average contents of EPA + DHA in their biomass from 2.3 to 9.67 mg g− 1

6
M.I. Gladyshev et al.
were reported (Saglik Aslan et al., 2007; Cladis et al., 2014; Neff et al.,
2014). Nevertheless, farmed rainbow trout were found to have EPA +
DHA contents from 2.4 to 21.1 mg g− 1 (Saglik Aslan et al., 2007; Stone
et al., 2011; Turchini et al., 2011, 2018). Thus, according to our study
and reports of other authors (Turchini et al., 2018), farmed rainbow
trout can have the nutritive value even higher, than that of the wild-
caught rainbow trout.
Most studied strains in all three farms, including the outbred fish in
Farm 3, had nearly similar contents of EPA and DHA in their biomass.
Nevertheless, fish of some strains, Adler, Augustin and Kamloops tended
to have comparatively lower contents of EPA and DHA. The average
content of EPA + DHA in farmed rainbow trout 2.03 ± 0.05 mg g− 1,
found in this study, was slightly lower, than minimum values, reported
by some other authors (Saglik Aslan et al., 2007; Stone et al., 2011).
The three strains, mentioned above, had spawning in late summer
and autumn, in contrast to the other studied strains, which had
spawning in winter and spring (Fig. 5). The correlation analysis proved a
relation of EPA + DHA contents with the season of spawning. Probably,
this relation took place due to a gradual decrease of water temperature
in Farm 2 in August – March from 14.5–15.0 С to 7.8–9.5 С. Decreasing
◦ ◦
of water temperature is known to increase a period of incubation of eggs,
and Salmoniformes with a longer period of incubation of eggs have a
higher EPA and DHA contents (Artamonova et al., 2020). In any case,
the difference in LC-PUFA content between the strains of rainbow trout,
reared in Farm 2, demonstrated a significant genetic control of this
content. Thus, a potential of selective breeding of rainbow trout strains
with a high EPA and DHA contents is believed to be possible, like that of
Atlantic salmon (Bell et al., 2010; Horn et al., 2018).
In this study, we regard the content of EPA + DHA as the main in­
dicator of the nutritive value of fish for humans. Evidently, besides
lipids, including LC-PUFA, fish products are valuable source of other
nutrients, e.g., amino acids (proteins). Nevertheless, fish give only ca.
6% of all animal and plant protein consumed by humans (Tacon and
Metian, 2013), while EPA and DHA obtained from fish constitute more
than 97% of these essential nutrients in humans’ diet (Gladyshev et al.,
2015). Thereby, the peculiar nutritive value of fish evidently consist in
the LC-PUFA, rather than in other nutrients.
It also should be noted, that namely content of EPA and DHA in fish
biomass, measured in muss units (mg g− 1), rather than their level,
measured in the relative units (% of total FA) should be regarded as the
indicator of nutritive value. Expressing of LC-PUFA in the relative units
give erroneous results concerning the real quantity of the essential nu­
trients, which could be obtained by humans with a certain portion of this
or that fish product (Gladyshev et al., 2007; Huynh and Kitts, 2009;
Joordens et al., 2014; Turchini et al., 2018; Gladyshev and Sushchik,
2019).
In our study, marker fatty acids were revealed, which potentially
allow to differentiate wild and farmed rainbow trout. For instance, in the
wild fish, level of oleic acid, 18:1n-9 was significantly lower, than that in
the farmed fish: 13.73 ± 1.10% (maximum 16.40%) and 29.69 ± 0.71%
(minimum 18.21%), respectively. Oleic acid is one of fatty acids that are
heavily catabolized for energy in fish (Tocher, 2003), and it was shown
to be one of the preferred lipid fuels in swimming muscles (McKenzie
et al., 1998). Farmed trout, limited in their swimming, evidently have
significantly lower energy demands compared to the wild trout. Thus,
farmed trout preferentially accumulated ‘energetic’ oleic acid in storage
lipids, rather than used it for catabolism. In other works, compared FA
composition of wild and farmed rainbow trout the same tendency could
be noticed: levels of 18:1n-9 in wild fish, 10.66–24.63%, were lower
than those in farmed fish, 22.9–30.47%, (Fallah et al., 2011; Ozogul
et al., 2013; Akpinar et al., 2015; Ural et al., 2017, but see Saglik Aslan
et al., 2007).
In last decades, vegetable oils are increasingly added to farmed fish
food (Turchini et al., 2009; Shepherd and Jackson, 2013; Sprague et al.,
2016; Qian et al., 2020; Barry and Trushenski, 2020; Quiñones et al.,
2021). It is not at the least surprising, that level of the other potential
7
Aquaculture 556 (2022) 738265
marker, 18:2n-6, which is one of the main constituent of vegetable oils
(Naylor et al., 2009; Hixson et al., 2014), in our study was significantly
higher in the farmed trout, than that in the wild trout: 11.47 ± 0.32%
(minimum 6.17%) and 1.94 ± 0.09% (maximum 2.25%), respectively.
Similarly, in other studies levels of 18:2n-6 in farmed rainbow trout,
6.2–17.15%, were higher than those in wild rainbow trout, 3.63–5.97%,
(Blanchet et al., 2005; Fallah et al., 2011; Ural et al., 2017, but see
Ozogul et al., 2013; Akpinar et al., 2015). In contrast, levels of another
potential marker FA, 20:5n-3, in our study was significantly higher in
the wild trout, than that in the farmed trout: 8.60 ± 0.44% (minimum
7.59%) and 4.11 ± 0.20% (maximum 7.85%), respectively. The same
tendency could be noticed in the other studies: levels of 20:5n-3 in wild
rainbow trout, 2.55–18.56%, were higher than those in farmed rainbow
trout, 1.77–10.29%, (Blanchet et al., 2005; Fallah et al., 2011; Ozogul
et al., 2013; Akpinar et al., 2015; Ural et al., 2017). Thus, levels of 18:1n-
9, 18:2n-6 and 20:5n-3 can be used as marker FA to differentiate wild
and farmed rainbow trout, but quantitative ranges for these markers
should be specified for each region.
5. Conclusions
As found, rainbow trout of strain Steelhead had comparatively
higher ability to regulate their FA composition and thereby to retain the
high contents of EPA and DHA in biomass despite variations of their
contents in diet. Thus, this strain is believed to be more suitable for
rearing on feeds with a high proportion of vegetable oils. In contrast,
trout of strains Adler, Augustin and Kamloops tended to have lower
contents of EPA + DHA than fish of the other strains, which is probably
caused by their spawning in late summer and autumn, in contrast to the
other strains with spawning in winter and spring.
Differences in contents of EPA + DHA of different strains, reared in
the same farm, are believed to prove a potential of selective breeding of
rainbow trout strains with a high EPA and DHA.
Wild and farmed rainbow trout can be differentiated using FA
composition: farmed fish had 18:2n-6 more than 6% and wild fish had
less than 6% of this FA in their flesh.
CRediT authorship contribution statement
Michail I. Gladyshev: Conceptualization, Data curation, Formal
analysis, Funding acquisition, Investigation, Methodology, Project
administration, Resources, Software, Visualization, Writing – original
draft, Writing – review & editing. Alexander A. Makhrov: Conceptu­
alization, Investigation, Data collection & curation, Writing – review &
editing. Nadezhda N. Sushchik: Investigation, Data collection &
curation, Writing – review & editing. Olesia N. Makhutova: Investi­
gation, Writing – review & editing. Anastasia E. Rudchenko: Investi­
gation, Writing – review & editing. Dmitrii A. Balashov: Investigation.
Evgenii V. Vinogradov: Investigation. Valentina S. Artamonova:
Conceptualization, Investigation, Data collection & curation, Writing –
review & editing.
Declaration of Competing Interest
No conflict of interest.
Acknowledgements
The work was supported by Federal Tasks for Institute of Biophysics
SB RAS No. 0287-2021-0019, Federal Tasks for Siberian Federal Uni­
versity No. FSRG-2020-0019, Federal Tasks for A. N. Severtzov Institute
of Ecology and Evolution RAS No. 0089-2021-0006 (АААА-А18-
118042490059-5) and by grant of the Krasnoyarsk Regional Science
Foundation No. 2021020207159 “Development of import-substituting
technologies for salmon aquaculture in the conditions of the Kras­
noyarsk Territory”.
M.I. Gladyshev et al. Aquaculture 556 (2022) 738265

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23.

8
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place them on a new theoretical basis. That basis, in accordance
with the general advance of thought, was supplied by religion.
Sexual relations which had once been condemned as wrong and
unnatural because they were supposed to thwart the natural
multiplication of animals and plants and thereby to diminish the food
supply, would now be condemned because it was imagined that they
were displeasing to gods or spirits, those stalking-horses which
savage man rigs out in the cast-off clothes of his still more savage
ancestors. The moral practice would therefore remain the same,
though its theoretical basis had been shifted from magic to religion.
In this or some such way as this we may conjecture that the Karens,
Dyaks, and other savages reached those curious conceptions of
sexual immorality and its consequences which we have been
considering. But from the nature of the case the development of
moral theory which I have sketched is purely hypothetical and hardly
admits of verification.
However, even if we assume for a moment that
But the reason why the savages in question reached their present
savages came to
regard certain view of sexual immorality in the way I have
sexual relations as surmised, there still remains the question, How did
irregular and they originally come to regard certain relations of
immoral remains
obscure. the sexes as immoral? For clearly the notion that
such immorality interferes with the course of
nature must have been secondary and derivative: people must on
independent grounds have concluded that certain relations between
men and women were wrong and injurious before they extended the
conclusion by false analogy to nature. The question brings us face to
face with the deepest and darkest problem in the history of society,
the problem of the origin of the laws which still regulate marriage and
the relations of the sexes among civilized nations; for broadly
speaking the fundamental laws which we recognize in these matters
are recognized also by savages, with this difference, that among
many savages the sexual prohibitions are far more numerous, the
horror excited by breaches of them far deeper, and the punishment
inflicted on the offenders far sterner than with us. The problem has
often been attacked, but never solved. Perhaps it is destined, like so
many riddles of that Sphinx which we call nature, to remain for ever
insoluble. At all events this is not the place to broach so intricate and
profound a discussion. I return to my immediate subject.
In the opinion of many savages the effect of
Sexual immorality is sexual immorality is not merely to disturb, directly
thought by many
savages to injure or indirectly, the course of nature by blighting the
the delinquents crops, causing the earth to quake, volcanoes to
themselves, their vomit fire, and so forth: the delinquents
offspring, and their
innocent spouses. themselves, their offspring, or their innocent
spouses are supposed to suffer in their own
persons for the sin that has been committed. Thus among the
Baganda of Central Africa “adultery was also regarded as a danger
to children; it was thought that women who were guilty of it during
pregnancy caused the child to die, either prior to birth, or at the time
of birth. Sometimes the guilty woman would herself die in childbed;
or, if she was safely delivered, she would have a tendency to devour
her child, and would have to be guarded lest she should kill it.”103.1
“When there was a case of retarded delivery, the relatives attributed
it to adultery; they made the woman confess the name of the man
with whom she had had intercourse, and if she died, her husband
was fined by the members of her clan, for they said: ‘We did not give
our daughter to you for the purpose of adultery, and you should have
guarded her.’ In most cases, however, the medicine-men were able
to save the woman’s life, and upon recovery she was upbraided, and
the man whom she accused was heavily fined.”103.2 The Baganda
thought that the infidelity of the father as well as of the mother
endangered the life of the child. For “it was also supposed that a
man who had sexual intercourse with any woman not his wife, during
the time that any one of his wives was nursing a child, would cause
the child to fall ill, and that unless he confessed his guilt and
obtained from the medicine-man the necessary remedies to cancel
the evil results, the child would die.”103.3 The common childish
ailment which was thought to be caused by the adultery of the father
or mother was called amakiro, and its symptoms were well
recognized: they consisted of nausea and general debility, and the
only cure for them was a frank confession by the guilty parent and
the performance of a magical ceremony by the medicine-man.103.4
 Similar views as to the disastrous effects of
Disastrous effects adultery on mother and child seem to be
of adultery on
adulteress and her widespread among Bantu tribes. Thus among the
child. Awemba of Northern Rhodesia, when both mother
and child die in childbirth, great horror is
expressed by all, who assert that the woman must assuredly have
committed adultery with many men to suffer such a fate. They exhort
her even with her last breath to name the adulterer; and whoever is
mentioned by her is called the “murderer” (musoka) and has
afterwards to pay a heavy fine to the injured husband. Similarly if the
child is born dead and the mother survives, the Awemba take it for
granted that the woman has been unfaithful to her husband, and
they ask her to name the murderer of her child, that is, the man
whose guilty love has been the death of the babe.104.1 In like manner
the Thonga, a Bantu tribe of South Africa, about Delagoa Bay, are of
opinion that if a woman’s travail pangs are unduly prolonged or she
fails to bring her offspring to the birth, she must certainly have
committed adultery, and they insist upon her making a clean breast
as the only means of ensuring her delivery; should she suppress the
name even of one of several lovers with whom she may have gone
astray, the child cannot be born. So convinced are the women of the
sufferings which adultery, if unacknowledged, entails on the guilty
mother in childbed, that a woman who knows her child to be
illegitimate will privately confess her sin to the midwife before she is
actually brought to bed, in the hope thereby of alleviating and
shortening her travail pangs.104.2 Further, the
Sympathetic
relation between an
Thonga believe that adultery establishes a
adulterer and the physical relationship of mutual sympathy between
injured husband. the adulterer and the injured husband such that
the life of the one is in a manner bound up with the
life of the other; indeed this relationship is thought to arise between
any two men who have had sexual connexion with the same woman.
As a native put it to a missionary, “They have met together in one life
through the blood of that woman; they have drunk from the same
pool.” To express it otherwise, they have formed a blood covenant
with each other through the woman as intermediary. “This
establishes between them a most curious mutual dependence:
should one of them be ill, the other must not visit him; the patient
might die. If he runs a thorn into his foot, the other must not help him
to extract it. It is taboo. The wound would not heal. If he dies, his
rival must not assist at his mourning or he would die himself.” Hence
if a man has committed adultery, as sometimes happens, with one of
his father’s younger wives, and the father dies, his undutiful son may
not take the part which would otherwise fall to him in the funeral
rites; indeed should he attempt to attend the burial, his relations
would drive him away in pity, lest by this mark of respect and
perhaps of remorse he should forfeit his life.105.1 In
Injurious effects of
adultery on the
like manner the Akikuyu of British East Africa
innocent husband, believe that if a son has adulterous intercourse
wife, or child. with one of his father’s wives, the innocent father,
not the guilty young scapegrace, contracts a
dangerous pollution (thahu), the effect of which is to make him ill and
emaciated or to break out into sores or boils, and even in all
probability to die, if the danger is not averted by the timely
intervention of a medicine-man.105.2 The Anyanja of British Central
Africa believe that if a man commits adultery while his wife is with
child, she will die; hence on the death of his wife the widower is often
roundly accused of having killed her by his infidelity.105.3 Without
going so far as this, the Masai of German East Africa hold that if a
father were to touch his infant on the day after he had been guilty of
adultery, the child would fall sick.105.4 According to the Akamba of
British East Africa, if a woman after giving birth to a child is false to
her husband before her first menstruation, the child will surely
die.105.5 The Akamba are also of opinion that if a
Injurious effects of
incest on the
woman is guilty of incest with her brother she will
offspring. be unable to bring to the birth the seed which she
has conceived by him. In that case the man must
purge his sin by bringing a big goat to the elders, and the woman is
ceremonially smeared with the contents of the animal’s stomach.106.1
Among the Washamba of German East Africa it happened that a
married woman lost three children, one after the other, by death. A
diviner being called in to ascertain the cause of this calamity,
attributed it to incest of which she had been accidentally guilty with
her father.106.2
Again, it appears to be a common notion with
Wife’s infidelity at savages that the infidelity of a wife prevents her
home thought to
endanger the husband from killing game, and even exposes him
absent husband in to imminent risk of being himself killed or wounded
the chase or the by wild beasts. This belief is entertained by the
war.
Wagogo and other peoples of East Africa, by the
Moxos Indians of Bolivia, and by Aleutian hunters of sea-otters. In
such cases any mishap that befalls the husband during the chase is
set down by him to the score of his wife’s misconduct at home; he
returns in wrath and visits his ill-luck on the often innocent object of
his suspicions even, it may be, to the shedding of her blood.106.3
While the Huichol Indians of Mexico are away seeking for a species
of cactus which they regard as sacred, their women at home are
bound to be strictly chaste; otherwise they believe that they would be
visited with illness and would endanger the success of the men’s
expedition.106.4 An old writer on Madagascar tells us that though
Malagasy women are voluptuous they will not allow themselves to be
drawn into an intrigue while their husbands are absent at the wars,
for they believe that infidelity at such a time would cause the absent
spouse to be wounded or slain.106.5 The Baganda of Central Africa
held similar views as to the fatal effect which a wife’s adultery at
home might have on her absent husband at the wars; they thought
that the gods resented her misconduct and withdrew their favour and
protection from her warrior spouse, thus punishing the innocent
instead of the guilty. Indeed, it was believed that if a woman were
even to touch a man’s clothing while her husband was away with the
army, it would bring misfortune on her husband’s weapon, and might
even cost him his life. The gods of the Baganda were most particular
about women strictly observing the taboos during their husbands’
absence and having nothing to do with other men all that time. On
his return from the war a man tested his wife’s fidelity by drinking
water from a gourd which she handed to him before he entered his
house. If she had been unfaithful to him during his absence, the
water was supposed to make him ill; hence should it chance that he
fell sick after drinking the draught, his wife was at once clapped into
the stocks and tried for adultery; and if she confessed her guilt and
named her paramour, the offender was heavily fined or even put to
death.107.1 Similarly among the Bangala or the Boloki of the Upper
Congo, “when men went to fight distant towns their wives were
expected not to commit adultery with such men as were left in the
town, or their husbands would receive spear wounds from the
enemy. The sisters of the fighters would take every precaution to
guard against the adultery of their brothers’ wives while they were on
the expedition.”107.2 So among the Haida Indians of the Queen
Charlotte Islands, while the men were away at the wars, their wives
“all slept in one house to keep watch over each other; for, if a woman
were unfaithful to her husband while he was with a war-party, he
would probably be killed.”107.3 If only King David had held this belief
he might have contented himself with a single instead of a double
crime, and need not have sent his Machiavellian order to put the
injured husband in the forefront of the battle.107.4
The Zulus imagine that an unfaithful wife who
Injurious effect of touches her husband’s furniture without first eating
wife’s infidelity on
her husband. certain herbs causes him to be seized with a fit of
coughing of which he soon dies. Moreover, among
the Zulus “a man who has had criminal intercourse with a sick
person’s wife is prohibited from visiting the sick-chamber; and, if the
sick person is a woman, any female who has committed adultery
with her husband must not visit her. They say that, if these visits ever
take place, the patient is immediately oppressed with a cold
perspiration and dies. This prohibition was thought to find out the
infidelities of the women and to make them fear discovery.”108.1 For a
similar reason, apparently, during the sickness of a
African chiefs Caffre chief his tribe was bound to observe strict
thought to be
injuriously affected continence under pain of death.108.2 The notion
by the incontinence seems to have been that any act of incontinence
of their subjects.
would through some sort of magical sympathy
prove fatal to the sick chief. The Ovakumbi, a tribe in the south of
Angola, think that the carnal intercourse of young people under the
age of puberty would cause the king to die within the year, if it were
not severely punished. The punishment for such a treasonable
offence used to be death.108.3 Similarly, in the kingdom of Congo,
when the sacred pontiff, called the Chitomé, was going his rounds
throughout the country, all his subjects had to live strictly chaste, and
any person found guilty of incontinence at such times was put to
death without mercy. They thought that universal chastity was
essential to the preservation of the life of the pontiff, whom they
revered as the head of their religion and their common father.
Accordingly when he was abroad he took care to warn his faithful
subjects by a public crier, that no man might plead ignorance as an
excuse for a breach of the law.108.4
Speaking of the same region of West Africa, an
Injurious effects of old writer tells us that “conjugal chastity is
adultery on the
adulteress. singularly respected among these people; adultery
is placed in the list of the greatest crimes. By an
opinion generally received, the women are persuaded that if they
were to render themselves guilty of infidelity, the greatest
misfortunes would overwhelm them, unless they averted them by an
avowal made to their husbands, and in obtaining their pardon for the
injury they might have done.”109.1 The Looboos of
Dangerous pollution
supposed to be
Sumatra think that an unmarried young woman
incurred by who has been got with child falls thereby into a
unchastity. dangerous state called looï, which is such that she
spreads misfortune wherever she goes. Hence
when she enters a house, the people try to drive her out by
force.109.2 Amongst the Sulka of New Britain unmarried people who
have been guilty of unchastity are believed to contract thereby a fatal
pollution (sle) of which they will die, if they do not confess their fault
and undergo a public ceremony of purification. Such persons are
avoided: no one will take anything at their hands: parents point them
out to their children and warn them not to go near them. The
infection which they are supposed to spread is apparently physical
rather than moral in its nature; for special care is taken to keep the
paraphernalia of the dance out of their way, the mere presence of
persons so polluted being thought to tarnish the paint on the
instruments. Men who have contracted this dangerous taint rid
themselves of it by drinking sea-water mixed with shredded coco-nut
and ginger, after which they are thrown into the sea. Emerging from
the water they put off the dripping clothes which they wore during
their state of defilement and cast them away. This purification is
believed to save their lives, which otherwise must have been
destroyed by their unchastity.109.3 Among the Buduma of Lake Chad,
in Central Africa, at the present day “a child born out of wedlock is
looked on as a disgrace, and must be drowned. If this is not done,
great misfortunes will happen to the tribe. All the men will fall sick,
and the women, cows and goats will become barren.”110.1
These examples may suffice to shew that
Conclusion. among many races sexual immorality, whether in
the form of adultery, fornication, or incest, is
believed of itself to entail, naturally and inevitably, without the
intervention of society, most serious consequences not only on the
culprits themselves, but also on the community, often indeed to
menace the very existence of the whole people by destroying the
food supply. I need hardly remind you that all these beliefs are
entirely baseless; no such consequences flow from such acts; in
short, the beliefs in question are a pure superstition. Yet we cannot
doubt that wherever this superstition has existed it must have served
as a powerful motive to deter men from adultery, fornication, and
incest. If that is so, then I think I have proved my third proposition,
which is, that among certain races and at certain times superstition
has strengthened the respect for marriage, and has thereby
contributed to the stricter observance of the rules of sexual morality
both among the married and the unmarried.
 V.
RESPECT FOR HUMAN LIFE

I pass now to my fourth and last proposition, which

Superstition as a is, that among certain races and at certain times
prop to the security
of human life. superstition has strengthened the respect for
human life and has thereby contributed to the
security of its enjoyment.
The particular superstition which has had this
The fear of ghosts. salutary effect is the fear of ghosts, especially the
ghosts of the murdered. The fear of ghosts is
widespread, perhaps universal, among savages; it is hardly extinct
among ourselves. If it were extinct, some learned societies might put
up their shutters. Dead or alive, the fear of ghosts has certainly not
been an unmixed blessing. Indeed it might with some show of
reason be maintained that no belief has done so much to retard the
economic and thereby the social progress of mankind as the belief in
the immortality of the soul; for this belief has led race after race,
generation after generation, to sacrifice the real wants of the living to
the imaginary wants of the dead. The waste and destruction of life
and property which this faith has entailed are enormous and
incalculable. Without entering into details I will
Disastrous illustrate by a single example the disastrous
consequences
entailed by the fear economic, political, and moral consequences
of the dead. which flow from that systematic destruction of
property which the fear of the dead has imposed
on many races. Speaking of the Patagonians, the well-informed and
intelligent traveller d’Orbigny observes: “They have no laws, no
punishments inflicted on the guilty. Each lives as he pleases, and the
greatest thief is the most highly esteemed, because he is the most
dexterous. A motive which will always prevent them from
abandoning the practice of theft, and at the same time will always
present an obstacle to their ever forming fixed settlements, is the
religious prejudice which, on the death of one of their number,
obliges them to destroy his property. A Patagonian, who has
amassed during the whole of his life an estate by thieving from the
whites or exchanging the products of the chase with neighbouring
tribes, has done nothing for his heirs; all his savings are destroyed
with him, and his children are obliged to rebuild their fortunes afresh,
—a custom which, I may observe in passing, is found also among
the Tamanaques of the Orinoco, who ravage the field of the
deceased and cut down the trees which he has planted;112.1 and
among the Yuracares, who abandon and shut up the house of the
dead, regarding it as a profanation to gather a single fruit from the
trees of his field. It is easy to see that with such customs they can
nourish no real ambition since their needs are limited to themselves;
it is one of the causes of their natural indolence and is a motive
which, so long as it exists, will always impede the progress of their
civilization. Why should they trouble themselves about the future
when they have nothing to hope from it? The present is all in all in
their eyes, and their only interest is individual; the son will take no
care of his father’s herd, since it will never come into his possession;
he busies himself only with his own affairs and soon turns his
thoughts to looking after himself and getting a livelihood. This
custom has certainly something to commend it from the moral point
of view in so far as it destroys all the motives for that covetousness
in heirs which is too often to be seen in our cities. The desire or the
hope of a speedy death of their parents cannot exist, since the
parents leave absolutely nothing to their children; but on the other
hand, if the Patagonians had preserved hereditary properties, they
would without doubt have been to-day in possession of numerous
herds, and would necessarily have been more formidable to the
whites, since their power in that case would have been more than
doubled, whereas their present habits will infallibly leave them in a
stationary state, from which nothing but a radical change will be able
to deliver them.”113.1 Thus poverty, indolence, improvidence, political
weakness, and all the hardships of a nomadic life are the miserable
inheritance which the fear of the dead entails on these wretched
Indians. Heavy indeed is the toll which superstition exacts from all
who pass within her gloomy portal.
 But I am not here concerned with the disastrous and deplorable
consequences, the unspeakable follies and crimes
Fear of the ghosts and miseries, which have flowed in practice from
of the slain a check the theory of a future life. My business at present
on murder.
is with the more cheerful side of the subject, with
the wholesome, though groundless, terror which ghosts, apparitions,
and spectres strike into the breasts of hardened ruffians and
desperadoes. So far as such persons reflect at all and regulate their
passions by the dictates of prudence, it seems plain that a fear of
ghostly retribution, of the angry spirit of their victim, must act as a
salutary restraint on their disorderly impulses; it must reinforce the
dread of purely secular punishment and furnish the choleric and
malicious with a fresh motive for pausing before they imbrue their
hands in blood. This is so obvious, and the fear of ghosts is so
notorious, that both might perhaps be taken for granted, especially at
this late hour of the evening. But for the sake of completeness I will
mention a few illustrative facts, taking them almost at random from
distant races in order to indicate the wide diffusion of this particular
superstition. I shall try to shew that while all ghosts are feared, the
ghosts of slain men are especially dreaded by their slayers.
The ancient Greeks believed that the soul of any
Ancient Greek man who had just been killed was angry with his
belief as to the
anger of a ghost at slayer and troubled him; hence even an
his slayer. involuntary homicide had to depart from his
country for a year until the wrath of the dead man
had cooled down; nor might the slayer return until sacrifice had been
offered and ceremonies of purification performed. If his victim
chanced to be a foreigner, the homicide had to shun the country of
the dead man as well as his own.114.1 The legend of the matricide
Orestes, how he roamed from place to place pursued and maddened
by the ghost of his murdered mother, reflects faithfully the ancient
Greek conception of the fate which overtakes the murderer at the
hands of the ghost.114.2
But it is important to observe that not only does
Among the Greeks the hag-ridden homicide go in terror of his victim’s
a manslayer was
dreaded and ghost; he is himself an object of fear and aversion
shunned because to the whole community on account of the angry
he was thought to and dangerous spirit which dogs his steps. It was
be haunted by the
angry and probably more in self-defence than out of
dangerous ghost of consideration for the manslayer that Attic law
his victim. compelled him to quit the country. This comes out
clearly from the provisions of the law. For in the
first place, on going into banishment the homicide had to follow a
prescribed road:114.3 obviously it would have been hazardous to let
him stray about the country with a wrathful ghost at his heels. In the
second place, if another charge was brought against a banished
homicide, he was allowed to return to Attica to plead in his defence,
but he might not set foot on land; he had to speak from a ship, and
even the ship might not cast anchor or put out a gangway. The
judges avoided all contact with the culprit, for they judged the case
sitting or standing on the shore.114.4 Plainly the intention of this rule
was literally to insulate the slayer, lest by touching Attic earth even
indirectly through the anchor or the gangway he should blast it by a
sort of electric shock, as we might say; though doubtless the Greeks
would have said that the blight was wrought by contact with the
ghost, by a sort of effluence of death. For the same reason if such a
man, sailing the sea, happened to be wrecked on the coast of the
country where his crime had been committed, he was allowed to
camp on the shore till a ship came to take him off, but he was
expected to keep his feet in sea-water all the time,115.1 evidently to
neutralise the ghostly infection and prevent it from spreading to the
soil. For the same reason, when the turbulent people of Cynaetha in
Arcadia had perpetrated a peculiarly atrocious massacre and had
sent envoys to Sparta, all the Arcadian states through which the
envoys took their way ordered them out of the country; and after
their departure the Mantineans purified themselves and their
belongings by sacrificing victims and carrying them round the city
and the whole of their land.115.2 So when the Athenians had heard of
a massacre at Argos, they caused purificatory offerings to be carried
round the public assembly.115.3
No doubt the root of all such observances was a
The legend of fear of the dangerous ghost which haunts the
Orestes reflects the
Greek horror of a murderer and against which the whole community
manslayer.
 as well as the homicide himself must be on its
guard. The Greek practice in these respects is clearly mirrored in the
legend of Orestes; for it is said that the people of Troezen would not
receive him in their houses until he had been purified of his guilt,115.4
that is, until he had been rid of his mother’s ghost. The Akikuyu of
British East Africa think that if a man who has killed another comes
and sleeps at a village and eats with a family in their hut, the persons
with whom he has eaten contract a dangerous pollution which might
prove fatal to them were it not removed in time by a medicine-man.
The very skin on which the homicide slept has absorbed the taint
and might infect any one else who slept on it. So a medicine-man is
sent for to purify the hut and its occupants.115.5
Manslayers purged
of the stain of
The Greek mode of purifying a homicide was to kill
human blood by a sucking pig and wash the hands of the guilty
being smeared with man in its blood: until this ceremony had been
the blood of pigs.
performed the manslayer was not allowed to
speak. 116.1 Among the hill-tribes near Rajamahal in Bengal, if two
men quarrel and blood be shed, the one who cut the other is fined a
hog or a fowl, “the blood of which is sprinkled over the wounded
person, to purify him, and to prevent his being possessed by a
devil.”116.2 In this case the blood-sprinkling is avowedly intended to
prevent the man from being haunted by a spirit; only it is not the
aggressor but his victim who is supposed to be in danger and
therefore to stand in need of purification. We have seen that among
these and other savage tribes pig’s blood is sprinkled on persons
and things as a mode of purifying them from the pollution of sexual
crimes.116.3 Among the Cameroon negroes in West Africa accidental
homicide can be expiated by the blood of an animal. The relations of
the slayer and of the slain assemble. An animal is killed, and every
person present is smeared with its blood on his face and breast.
They think that the guilt of manslaughter is thus atoned for, and that
no punishment will overtake the homicide.116.4 In Car Nicobar a man
possessed by devils is cleansed of them by being rubbed all over
with pig’s blood and beaten with leaves. The devils are supposed to
be thus swept off like flies from the man’s body to the leaves, which
are then folded up and tied tightly with a special kind of string. A
professional exorciser administers the beating, and at every stroke
with the leaves he falls down with his face on the floor and calls out
in a squeaky voice, “Here is a devil.” This ceremony is performed by
night; and before daybreak all the packets of leaves containing the
devils are thrown into the sea.117.1 The Greeks similarly used laurel
leaves as well as pig’s blood in purificatory ceremonies.117.2 In all
such cases we may assume that the purification was originally
conceived as physical rather than as moral, as a sort of detergent
which washed, swept, or scraped the ghostly or demoniacal pollution
from the person of the ghost-haunted or demon-possessed man.
The motive for employing blood in these rites of cleansing is not
clear. Perhaps the purgative virtue ascribed to it may have been
based on the notion that the offended spirit accepts the blood as a
substitute for the blood of the man or woman.117.3 However, it is
doubtful whether this explanation could cover all the cases in which
blood is sprinkled as a mode of purification. Certainly it is odd, as the
sage Heraclitus long ago remarked, that blood-stains should be
thought to be removed by blood-stains, as if a man who had been
bespattered with mud should think to cleanse himself by
bespattering himself with more mud.117.4 But the ways of man are
wonderful and sometimes past finding out.
There was a curious story that after Orestes had
The matricide gone mad through murdering his mother he
Orestes is said to
have recovered his recovered his wits by biting off one of his own
wits by biting off fingers; the Furies of his murdered mother, which
one of his own had appeared black to him before, appeared white
fingers.
as soon as he had mutilated himself in this way: it
was as if the taste of his own blood sufficed to avert or disarm the
wrathful ghost.117.5 A hint of the way in which the blood may have
been supposed to produce this result is furnished by the practice of
some savages. The Indians of Guiana believe that
Manslayers an avenger of blood who has slain his man must
commonly taste
their victims’ blood go mad unless he tastes the blood of his victim;
in order not to be the notion apparently is that the ghost drives him
haunted by their crazy, just as the ghost of Clytemnestra did to
ghosts.
Orestes, who was also, be it remembered, an
avenger of blood. In order to avert this consequence the Indian
manslayer resorts on the third night to the grave of his victim, pierces
the corpse with a sharp-pointed stick, and withdrawing it sucks the
blood of the murdered man. After that he goes home with an easy
mind, satisfied that he has done his duty and that he has nothing
more to fear from the ghost.118.1 A similar custom was observed by
the Maoris in battle. When a warrior had slain his foe in combat, he
tasted his blood, believing that this preserved him from the avenging
spirit (atua) of his victim; for they imagined that “the moment a slayer
had tasted the blood of the slain, the dead man became a part of his
being and placed him under the protection of the atua or guardian-
spirit of the deceased.”118.2 Thus in the opinion of these savages, by
swallowing a portion of their victim they made him a part of
themselves and thereby converted him from an enemy into an ally;
they established, in the strictest sense of the words, a blood-
covenant with him. The Aricara Indians also drank the blood of their
slain foes and proclaimed the deed by the mark of a red hand on
their faces.118.3 The motive for this practice may have been, as with
the Maoris, a desire to appropriate and so disarm the ghost of an
enemy. In antiquity some of the Scythians used to drink the blood of
the first foes they killed; and they also tasted the blood of the friends
with whom they made a covenant, for “they take that to be the surest
pledge of good faith.”118.4 The motive of the two
Homicides
supposed to go
customs was probably the same. “To the present
mad unless they day, when a person of another tribe has been slain
taste the blood of by a Nandi, the blood must be carefully washed off
their victim.
the spear or sword into a cup made of grass, and
drunk by the slayer. If this is not done it is thought that the man will
become frenzied.”118.5 So among some tribes of the Lower Niger “it
is customary and necessary for the executioner to lick the blood that
is on the blade”; moreover “the custom of licking the blood off the
blade of a sword by which a man has been killed in war is common
to all these tribes, and the explanation given me by the Ibo, which is
generally accepted, is, that if this was not done, the act of killing
would so affect the strikers as to cause them to run amok among
their own people; because the sight and smell of blood render them
absolutely senseless as well as regardless of all consequences. And
this licking the blood is the only sure remedy, and the only way in
which they can recover themselves.”119.1 So, too, among the Shans
of Burma “it was the curious custom of executioners to taste the
blood of their victims, as they believed if this were not done illness
and death would follow in a short time. In remote times Shan soldiers
always bit the bodies of men killed by them in battle.”119.2 Strange as
it may seem, this truly savage superstition exists apparently in Italy
to this day. There is a widespread opinion in Calabria that if a
murderer is to escape he must suck his victim’s blood from the
reeking blade of the dagger with which he did the deed.119.3 We can
now perhaps understand why the matricide Orestes was thought to
have recovered his wandering wits as soon as he had bitten off one
of his fingers. By tasting his own blood, which was also that of his
victim, since she was his mother, he might be supposed to form a
blood-covenant with the ghost and so to convert it from a foe into a
friend. The Kabyles of North Africa think that if a
Various precautions murderer leaps seven times over his victim’s grave
taken by
manslayers against within three or seven days of the murder, he will
the ghosts of their be quite safe. Hence the fresh grave of a
victims.
murdered man is carefully guarded.119.4 The
Lushai of North-Eastern India believe that if a man kills an enemy the
ghost of his victim will haunt him and he will go mad, unless he
performs a certain ceremony which will make him master of the dead
man’s soul in the other world. The ceremony includes the sacrifice of
an animal, whether a pig, a goat, or a mithan.120.1 Among the
Awemba of Northern Rhodesia, “according to a superstition common
among Central African tribes, unless the slayers were purified from
blood-guiltiness they would become mad. On the night of return no
warrior might sleep in his own hut, but lay in the open nsaka in the
village. The next day, after bathing in the stream and being anointed
with lustral medicine by the doctor, he could return to his own hearth,
and resume intercourse with his wife.”120.2 In all such cases the
madness of the slayer is probably attributed to the ghost of the slain,
which has taken possession of him.
 That the Greek practice of secluding and
The custom of purifying a homicide was essentially an exorcism,
secluding and
purifying homicidesin other words, that its aim was to ban the
is intended to dangerous ghost of his victim, is rendered
protect them practically certain by the similar rites of seclusion
against the angry
and purification which among many savage tribes
spirits of the slain,
which are thought have to be observed by victorious warriors with the
to madden their
slayers.
avowed intention of securing them against the
spirits of the men whom they have slain in battle.
These rites I have illustrated elsewhere,120.3 but a few cases may be
quoted here by way of example. Thus among the Basutos “ablution
is especially performed on return from battle. It is absolutely
necessary that the warriors should rid themselves, as soon as
possible, of the blood they have shed, or the shades of their victims
would pursue them incessantly, and disturb their slumbers. They go
in a procession, and in full armour, to the nearest stream. At the
moment they enter the water a diviner, placed higher up, throws
some purifying substances into the current.”120.4 According to
another account of the Basuto custom, “warriors who have killed an
enemy are purified. The chief has to wash them, sacrificing an ox in
the presence of the whole army. They are also anointed with the gall
of the animal, which prevents the ghost of the enemy from pursuing
them any farther.”121.1 Among the Thonga, a Bantu tribe of South
Africa, about Delagoa Bay, “to have killed an enemy on the battle-
field entails an immense glory for the slayers; but that glory is fraught
with great danger. They have killed.… So they are exposed to the
mysterious and deadly influence of the nuru and must consequently
undergo a medical treatment. What is the nuru? Nuru, the spirit of
the slain which tries to take its revenge on the slayer. It haunts him
and may drive him into insanity: his eyes swell, protrude and become
inflamed. He will lose his head, be attacked by giddiness
(ndzululwan) and the thirst for blood may lead him to fall upon
members of his own family and to stab them with his assagay. To
prevent such misfortunes, a special medication is required: the
slayers must lurulula tiyimpì ta bu, take away the nuru of their
sanguinary expedition.… In what consists this treatment? The
slayers must remain for some days at the capital. They are taboo.
They put on old clothes, eat with special spoons, because their
hands are ‘hot,’ and off special plates (mireko) and broken pots.
They are forbidden to drink water. Their food must be cold. The chief
kills oxen for them; but if the meat were hot it would make them swell
internally ‘because they are hot themselves, they are defiled (ba na
nsila).’ If they eat hot food, the defilement would enter into them.
‘They are black (ntima). This black must be removed.’ During all this
time sexual relations are absolutely forbidden to them. They must
not go home, to their wives. In former times the Ba-Ronga used to
tattoo them with special marks from one eyebrow to the other.
Dreadful medicines were inoculated in the incisions, and there
remained pimples ‘which gave them the appearance of a buffalo
when it frowns.’ After some days a medicine-man comes to purify
them, ‘to remove their black.’ There seem to be various means of
doing it, according to Mankhelu. Seeds of all kinds are put into a
broken pot and roasted, together with drugs and psanyi122.1 of a
goat. The slayers inhale the smoke which emanates from the pot.
They put their hands into the mixture and rub their limbs with it,
especially the joints.… Insanity threatening those who shed blood
might begin early. So, already on the battle-field, just after their deed,
warriors are given a preventive dose of the medicine by those who
have killed on previous occasions.… The period of seclusion having
been concluded by the final purification, all the implements used by
the slayers during these days, and their old garments, are tied
together and hung by a string to a tree, at some distance from the
capital, where they are left to rot.”122.2
The accounts of the madness which is apt to
With some savages befall slayers seem too numerous and too
temporary insanity
seems to be really consistent to be dismissed as pure fictions of the
caused by the sight savage imagination. However we may reject the
or even thought of native explanation of such fits of frenzy, the
blood.
reports point to a real berserker fury or unbridled
thirst for blood which comes over savages when they are excited by
combat, and which may prove dangerous to friends as well as to
foes. The question is one on which students of mental disease might
perhaps throw light. Meantime it deserves to be noticed that even
the people who have staid at home and have taken no share in the
bloody work are liable to fall into a state of frenzy when they hear the
war-whoops which proclaim the approach of the victorious warriors
with their ghastly trophies. Thus we are told that among the Bare’e-
speaking Toradjas of Central Celebes, when these notes of triumph
were heard in the distance the whole population of the village would
turn out to meet and welcome the returning braves. At the mere
sound some of those who had remained at home, especially women,
would be seized with a frenzy, and rushing forth would bite the
severed heads of the slain foes, and they were not to be brought to
their senses till they had drunk palm wine or water out of the skulls. If
the warriors returned empty-handed, these furies would fall upon
them and bite their arms. There was a regular expression for this
state of temporary insanity excited by the sight or even the thought
of human blood; it was called merata lamoanja or merata raoa, “the
spirit is come over them,” by which was probably meant that the
madness was caused by the ghosts of the slaughtered foes. When
any of the warriors themselves suffered from this paroxysm of frenzy,
they were healed by eating a piece of the brains or licking the blood
of the slain.123.1
Among the Bantu tribes of Kavirondo, in British
Means taken by East Africa, when a man has killed an enemy in
manslayers in
Africa to rid warfare he shaves his head on his return home,
themselves of the and his friends rub a medicine, which generally
ghosts of their consists of cow’s dung, over his body to prevent
victims.
the spirit of the slain man from troubling him.123.2
Here cow’s dung serves these negroes as a detergent of the ghost,
just as pig’s blood served the ancient Greeks. Among the Wawanga,
about Mount Elgon in British East Africa, “a man returning from a
raid, on which he has killed one of the enemy, may not enter his hut
until he has taken cow-dung and rubbed it on the cheeks of the
women and children of the village and purified himself by the
sacrifice of a goat, a strip of skin from the forehead of which he
wears round the right wrist during the four following nights.”123.3 With
the Ja-Luo of Kavirondo the custom is somewhat different. Three
days after his return from the fight the warrior shaves his head. But
before he may enter his village he has to hang a live fowl, head
uppermost, round his neck; then the bird is decapitated and its head
left hanging round his neck. Soon after his return a feast is made for
the slain man, in order that his ghost may not haunt his slayer.123.4 In
some of these cases the slayer shaves his head, precisely as the
matricide Orestes is said to have shorn his hair when he came to his
senses.123.5 From this Greek tradition we may infer with some
probability that the hair of Greek homicides, like that of these African
warriors, was regularly cropped as one way of ridding them of the
ghostly infection. Among the Ba-Yaka, a Bantu people of the Congo
Free State, “a man who has been killed in battle is supposed to send
his soul to avenge his death on the person of the man who killed
him; the latter, however, can escape the vengeance of the dead by
wearing the red tail-feathers of the parrot in his hair, and painting his
forehead red.”124.1 Perhaps, as I have suggested elsewhere, this
costume is intended to disguise the slayer from his victim’s
ghost.124.2 Among the Natchez Indians of North
Precautions taken
by the Natchez
America young braves who had taken their first
Indians. scalps were obliged to observe certain rules of
abstinence for six months. They might not sleep
with their wives nor eat flesh; their only food was fish and hasty-
pudding. If they broke these rules they believed that the soul of the
man they had killed would work their death by magic.124.3
The Kai of German New Guinea stand in great
Ghosts of the slain fear of the ghosts of the men whom they have
dreaded by the Kai
of German New slain in war. On their way back from the field of
Guinea. battle or the scene of massacre they hurry in order
to be safe at home or in the shelter of a friendly
village before nightfall; for all night long the spirits of the dead are
believed to dog the footsteps of their slayers, in the hope of coming
up with them and recovering the lost portions of their souls which
adhere with the clots of their blood to the spears and clubs that dealt
them the death-blow. Only so can these poor restless ghosts find
rest and peace. Hence the slayers are careful not to bring back the
blood-stained weapons with them into the village; for that would be
the first place where the ghosts would look for them. They hide them,
therefore, in the forest at a safe distance from the village, where the
ghosts can never find them; and when the spirits are weary of the