Co-cultivation of Isochrysis galbana

and Marinobacter sp. can enhance algal

growth and docosahexaenoic acid
production Ying-Ying Wang
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 Aquaculture 556 (2022) 738248

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Co-cultivation of Isochrysis galbana and Marinobacter sp. can enhance algal

growth and docosahexaenoic acid production
Ying-Ying Wang a, Si-Min Xu a, Jia-Yi Cao a, *, Min-Nan Wu a, Jing-Hao Lin a, Cheng-Xu Zhou a,
Lin Zhang a, Hai-Bo Zhou b, Yan-Rong Li d, Ji-Lin Xu a, *, Xiao-Jun Yan c
a
Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ministry of Education of China, Ningbo, Zhejiang 315211, China
b
Fujian Dalai Seed Science and Technology Co. LTD, Ningde, Fujian 352101, China
c
Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture, Ningbo University, Ningbo, Zhejiang 315211, China
d
Ningbo Institute of Oceanography, Ningbo, Zhejiang 315832, China

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

Keywords: Isochrysis galbana, an important diet microalgal species, is widely used in aquaculture and rich in docosahexa­
Isochrysis galbana enoic acid (DHA). Currently, no efficient strategies are available to steadily enhance the production of its biomass
Marinobacter sp. and DHA. It has been reported that algae-associated bacteria can affect the yield of algae and the production of
Growth-promoting bacteria
high-value compounds. Here, we identified a growth-promoting bacterial strain Marinobacter sp., which could
DHA
enhance the growth, the content of chlorophyll a, the maximal photochemical efficiency of PS II (Fv/Fm), and
soluble protein content of I. galbana when they were co-cultured, while superoxide dismutase (SOD) activity was
decreased. Besides, Marinobacter sp. promoted the production of DHA and up-regulated the expressions of genes
involved in DHA synthesis pathway of I. galbana. Our study clearly suggested that co-cultivation of I. galbana and
Marinobacter sp. could effectively enhance the quality and quantity of microalgae, showing promising applica­
tions in improving productivity and sustainability of aquaculture algal rearing systems.

1. Introduction cultivation of high-quality microalgae and improving their high-value

Microalgae have potential applications as food, medicine, health
products, biomass energy, industrial materials, bait in aquaculture and
so on (Harun et al., 2010; Hemaiswarya et al., 2011; Matos et al., 2017).
Microalgae are rich in nutrients, including proteins, amino acids, pig­
ments, carbohydrates, lipids, vitamins, and some minerals (Buono et al.,
2014; Matos et al., 2017; Pradhan et al., 2021). Among them, docosa­
hexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most
valuable functional components in microalgal lipids, which have been
widely believed to benefit human health (Romieu et al., 2005). Besides,
these microalgal nutrients have also been widely used in aquaculture.
Microalgae as bait or bait supplements can alleviate the barriers to fish
meal farming and have been proved to be a successful replacement for
fish meal (Dineshbabu et al., 2019). Some microalgae, such as Tha­
lassiosira pseudonana, are widely cultivated to feed the Pacific oyster
Crassostrea gigas and rock scallops (Yaakob et al., 2014). Therefore, the
substances are very important for aquaculture.
As one of the most important bait microalgae, I. galbana belongs to
the class of Prymnesiophyceae (Matos et al., 2019). Extensive research
has shown that I. galbana is widely used to feed bivalve larvae, such as
mussels, scallops, clams, and bivalve larvae (Dineshbabu et al., 2019;
Tremblay et al., 2007), because it is small in size, easy to be digested,
and rich in essential nutritional constituents, such as DHA and EPA. As a
potential algal strain with abundant DHA and EPA, it has been observed
that I. galbana is not only a good bait for aquaculture seedling but also an
important raw material for the development of bioactive substances (Liu
et al., 2013). Meanwhile, it is considered to have the potential to achieve
industrialization. Therefore, a great deal of attention has been paid to
the study of I. galbana recently.
To improve the wide application of I. galbana, it is of great signifi­
cance to enhance its biomass yield. Studies have found that the growth
of I. galbana is affected by many abiotic factors, such as light,

Abbreviations: DHA, docosahexaenoic acid; SOD, superoxide dismutase; EPA, eicosapentaenoic acid; BCA, bicinchoninic acid; FAMEs, Fatty acid methyl esters;
RT-qPCR, Real-time quantitative PCR; rbcL, ribulose-1,5-bisphosphate carboxylase/oxygenase; d4FAD, delta-4 desaturase gene; ASE2, delta-9 elongase gene; SFAs,
saturated fatty acids; MUFAs, monounsaturated fatty acids; ROS, reactive oxygen species; VF, vibrioferrin.
* Corresponding authors.
E-mail addresses: caojiayi@nbu.edu.cn (J.-Y. Cao), xujilin@nbu.edu.cn (J.-L. Xu).

https://doi.org/10.1016/j.aquaculture.2022.738248
Received 14 December 2021; Received in revised form 21 March 2022; Accepted 8 April 2022
Available online 11 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
Y.-Y. Wang et al.
temperature, salinity, CO2, and nutrients (Alkhamis and Qin, 2013; Cao
et al., 2020; Picardo et al., 2013; Sayegh and Montagnes, 2011; Zhang
et al., 2014). However, even if the main abiotic factors of the growth
environment of I. galbana are controlled under optimal conditions, there
are still many problems under large-scale outdoor conditions, reminding
us that the growth is also impaired by other factors in addition to the
above-mentioned abiotic factors. Algae will release a large number of
carbohydrates, amino acids, enzymes, lipids, and other metabolites into
the environment during the growth process to form a unique “phyco­
sphere”, which will attract a lot of bacteria-dominated microorganisms
(Bell and Mitchell, 1972). In this environment, the extracellular prod­
ucts of algae will stimulate bacterial growth and vice versa. Therefore,
the interactions between these two groups and the influence of their
interaction on each other are areas of recent research interest. Besides
the influence on growth, it has been reported that bacteria also affect the
production of high-value compounds of microalgae. For example,
Pseudomonas composti promoted the increase in biomass yield and lipid
of Characium sp. 46–4 by releasing some unidentified extracellular
compounds (Berthold et al., 2019). In addition, Liu et al. (2020) have
found that co-culture with probiotic algae-associated bacteria signifi­
cantly enhances the EPA production of Nannochloropsis oceanica. In the
case of I. galbana, Sandhya and Vijayan (2019) have shown that some
I. galbana-associated bacteria play a role in promoting algal growth.
Moreover, the interaction between them is regulated by various growth-
stimulating compounds, such as antioxidants, siderophores, and indole-
3-acetic acid, which can have a significant positive impact on algal
growth. However, the relationship between this microalgae and bacteria
remains largely unexplored. Therefore, it is urgently necessary to
explore the growth-promoting bacteria and their effects on the physi­
ology and metabolism of I. galbana, as well as the interaction mechanism
between them.
In the present study, we preliminarily applied different initial algae/
bacteria ratios to explore the effects of I. galbana-associated bacteria
Marinobacter sp. on the growth of I. galbana. We further evaluated the
physiological and biochemical effects of Marinobacter sp. on I. galbana.
Collectively, our current findings not only improved the understanding
of bacteria-microalgae interactions but also provided an alternative
strategy to improve algal production and culture stability.
2. Materials and methods
2.1. Culture of microalgae and bacteria
I. galbana 3011 was obtained from the Marine Biotechnology Labo­
ratory of Ningbo University, China. As a culture medium, the seawater
was filtered through 0.22-μm cellulose acetate membranes and then
sterilized by autoclaving. NMB3 medium used in this study was
composed of KNO3 (100 mg/L), KH2PO4 (10 mg/L), MnSO4⋅H2O (2.5
mg/L), FeSO4⋅7H2O (2.5 mg/L), EDTA-Na2 (10 mg/L), vitamin B1 (6 μg/
L), and vitamin B12 (0.05 μg/L) (Yang et al., 2016). A photoperiod of 12/
12-h day/night was applied with a light intensity of 100 μmol photon
m− 2 s− 1 using a cool light fluorescent lamp. The temperature was
controlled at 25 ◦ C throughout the experiment. Marinobacter sp. used in
this study was isolated from I. galbana culture and preserved in the
Marine Biotechnology Laboratory of Ningbo University. Marinobacter sp.
was freshly cultured on a 2216E marine medium at 28 ◦ C before
experiments.
2.2. Co-culture experiments
Before co-culture experiments, Marinobacter sp. was plated freshly on
a 2216E agar medium, and single colonies were grown in the 2216E
liquid medium overnight (25 ◦ C, 180 rpm). The freshly prepared bac­
terial culture broth was centrifuged at 5000 ×g for 5 min, washed twice,
and finally resuspended in sterile NMB3 medium. The axenic I. galbana
was obtained and maintained as previously described (Cao et al., 2019).
2
Aquaculture 556 (2022) 738248
When axenic I. galbana was cultured to an exponential phase (initial cell
density of about 1 × 106 cells/mL), the bacterial suspension was added
into the algal culture to achieve an algae/bacteria ratio of 1:1, 1:50, or
1:100 (cell counts: cell counts). Axenic I. galbana cultured alone was
denoted as the control group.
2.3. Physiological and biochemical determinations of microalgae
To study the physiological and biochemical effects of Marinobacter
sp. on I. galbana, the algae/bacteria ratio in the co-culture was 1:100.
The content of chlorophyll a, the maximal photochemical efficiency of
PS II (Fv/F), soluble protein content, and superoxide dismutase (SOD)
activity of I. galbana in the co-culture and mono-culture systems were
measured every 2 days. The content of chlorophyll a was measured as
previously described (Bruckner et al., 2008). The absorbance was
determined at wavelengths of 630, 664, and 750 nm. The content of
chlorophyll a was then calculated using the following equation:
Chl a (μg/mL) = 11.47 × (OD664 − OD750 ) − 0.4 × (OD630 − OD750 )
Fv/Fm was measured by AquaPen, which is a lightweight, hand-held
fluorometer intended for quick and reliable measurements of photo­
synthetic activity in algae. The measurement was performed with a
dark-adapted sample. Soluble protein was extracted and analyzed using
the bicinchoninic acid (BCA) protein assay with bovine serum albumin
as the standard according to the manufacturer’s instructions. The con­
centration of soluble protein was calculated based on the standard curve
drawn. The SOD activity was determined by the xanthine oxidase
method at a wavelength of 550 nm according to the manufacturer’s
instructions.
2.4. Fatty acid analysis
For fatty acid analysis, the samples from mono-culture of I. galbana,
mono-culture of Marinobacter sp., and their co-cultures were collected
on the 14th day post-co-culturing by centrifuging at 10,000 rpm for 10
min. The obtained I. galbana and Marinobancter sp. pellets were pre-
frozen in liquid nitrogen and dried by the vacuum freeze-drying sys­
tem for further analysis.
Fatty acid methyl esters (FAMEs) were purified following the
methods with modifications as previously described (Okuyama et al.,
1992). Briefly, samples (20 mg) of lyophilized cells were accurately
weighed and transferred to culture tubes. Next, 1 mL n-hexane, 15 μL
internal standard nonadecanoic acid (1 mg/mL), and 1.5 mL freshly
made pyromrcyl chloride (prepared by slowly adding 10 mL of acetyl
chloride to 100 mL of anhydrous methanol) were added to each sample
in a culture tube and vortexed for 1 min at a slow speed. The tightly
capped tubes were heated at 70 ◦ C in a water bath for 2 h. After samples
were cooled to room temperature, 2.5 mL of 6% K2CO3 was added,
followed by the addition of 1 mL n-hexane. The tubes were vortexed 30s
and the upper phase was collected in 2-mL sample bottle, then cen­
trifugated at 3000 rpm for 10 min. The supernatant was filtered by an
organic phase filter membrane, and the filtrate was collected into a 2-mL
screw-capped sample bottle. The purified fatty acids were subjected to
methylations.
The methyl esters were analyzed using an Agilent 7890B–7000C gas
chromatography system equipped with an EI detector. A 100-m-long
capillary column CD-2560 (Germany, CNW) was used. The column
had an inside diameter of 0.25 mm and a film thickness of 0.2 μm. The
temperature program was set as follows. The initial oven temperature
was maintained at 140 ◦ C for 5 min, then the temperature was increased
to 240 ◦ C at an increment of 4 ◦ C/min; and finally, the oven was
maintained isothermally for 30 min. The injector temperature was set at
250 ◦ C. Helium was used as the carrier gas at a constant flow rate of 2.25
mL/min. In each analysis, 1 μL of methyl ester solution was injected into
the chromatograph. Fatty acids were identified by comparison of
Y.-Y. Wang et al.
retention times to those of known standards and expressed as % of
FAMEs identified. The concentration of each fatty acid was calculated
using Qualitative Analysis, B.07.00 software.
2.5. Total RNA extraction
Microalgae were harvested through centrifugation at 10,000 rpm
and 4 ◦ C for 10 min. Algal cells were ground into powder in liquid ni­
trogen. Total RNA was isolated from each sample using an E. Z. N. A.®
Plant RNA Kit (OMEGA Bio-Tek) according to the manufacturer’s in­
structions. The concentration and quality of the purified RNA were
determined using a Thermo Scientific NanoDrop One spectrophotometer
(Thermo Fisher Scientific, USA).
2.6. Real-time quantitative PCR (RT-qPCR)
Briefly, purified RNA was reversely transcribed into cDNA using
PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time)
(TaKaRa, Japan). Gene expression was analyzed through RT-qPCR using
a LongGene Q2000A qPCR system (Hangzhou) and TB Greer® Premix
Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Japan). The large subunit gene
of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) was used as
the reference gene (Han et al., 2019). Primers for delta-4 desaturase
gene (d4FAD), d5FAD, d6FAD, and d8FAD were synthesized as previ­
ously reported (Huerlimann et al., 2014). The primers for the delta-9
elongase gene (ASE2) were designed using Primer 5. All the PCR
primers were listed in Table S1. The relative expressions of target genes
were calculated using the 2^-ΔΔCt method as previously described (Cao
et al., 2016).
3. Results
3.1. The effects of Marinobacter sp. on the growth of I. galbana
The axenic strain of I. galbana was cultured in the presence of Mar­
inobacter sp. to investigate the effect of Marinobacter sp. on microalgal
growth. Fig. 1 shows the effects of Marinobacter sp. with different algae/
bacteria ratios (1:1, 1:50, and 1:100) on the content of chlorophyll a of
I. galbana over a period of 14 days. When we explored the effects of
Marinobacter sp. on the growth of I. galbana, we found that Marinobacter
sp. exerted a growth-promoting effect on I. galbana depending on the
population density of the bacteria. Besides, the growth-promoting ef­
fects were enhanced with the increase of the treatment duration of
bacteria. The presence of bacteria under ratios of 1:50 and 1:100
significantly increased the cell growth of I. galbana compared with the
control group. We further detected the cell growth of these two strains
during the co-cultivation. The cell growth of I. galbana was enhanced by
Marinobacter sp., while the growth of Marinobacter sp. was decreased
when compared with pure culture of bacteria from day 4 (Fig. S1).
Moreover, no significant effect on the morphology of Marinobacter sp.
was found in co-cultivation. For I. galbana, at a later stage of the growth,
a large number of deformed algal cells were observed in the mono-
culture, while no deformation of algal cells was observed in the co-
cultures (Fig. S2).
3.2. The physiological and biochemical effects of Marinobacter sp. on
I. galbana
To study the physiological and biochemical effects of Marinobacter
sp. on I. galbana, the content of chlorophyll a, Fv/F, soluble protein
content, and SOD activity of I. galbana in the co-culture and mono-
culture systems were compared (Fig. 2). During 14 days of moni­
toring, the content of algal chlorophyll a in co-cultures was significantly
higher compared with the I. galbana mono-culture (Fig. 2A). The chlo­
rophyll fluorescence parameter, Fv/Fm, was used to monitor the effect of
Marinobacter sp. on the photosynthetic property of I. galbana. Fig. 2B
3
Aquaculture 556 (2022) 738248
Fig. 1. Co-culture results of Isochrysis galbana and Marinobacter sp. with three
different initial algae/bacteria ratios (1:1, 1:50, 1:100) expressed as chlorophyll
a content over the period of 14 days. Significance of the differences between
mean values was determined with Student’s t-test. All the experiments were
carried out in triplicate with standard error as error bar, while asterisks (*)
indicate significant difference at p value <0.05.
shows that Fv/Fm was higher in the co-culture system during the whole
culture process compared with the mono-culture. The results revealed
that the photosynthetic property of I. galbana was improved in the
presence of Marinobacter sp. The total concentration of soluble protein in
the microalgal cells was increased steadily over time (Fig. 2C), showing
that the presence of Marinobacter sp. significantly increased the syn­
thesis of soluble protein in I. galbana. The SOD activity in I. galbana was
decreased in the co-culture system compared with the mono-culture
(Fig. 2D). The presence of Marinobacter sp. inhibited the SOD activity
in microalgae during the entire study period. On the 8th day of the
experiment, the highest SOD activity was observed in the co-culture,
while it was still lower compared with the mono-culture at the corre­
sponding time point. The peak value in the mono-culture on the last day
was 1.27-fold higher compared with the co-culture group.
3.3. The effects of Marinobacter sp. on the fatty acid composition of
I. galbana
The difference in fatty acid composition of I. galbana in the co-culture
and mono-culture systems was analyzed (Table 1-3, Table S2). The most
abundant saturated fatty acids (SFAs) in both axenic and co-cultured
I. galbana were myristic acid (C14:0) and palmitic acid (C16:0). The
specific SFAs obtained from the mono-culture of Marinobacter sp. con­
sisted of dodecanoic acid (C12:0) and tridecanoic acid (C13:0). Addi­
tionally, the main SFAs in Marinobacter sp. were C12:0 (22.81 ± 0.66%)
and C16:0 (29.91 ± 1.03%). Moreover, we found that the contents of
pentadecanoic acid (C15:0), stearic acid (C18:0), and behenic acid
(C22:0) were significantly decreased in I. galbana when co-cultured with
Marinobacter sp. The levels of total monounsaturated fatty acids
(MUFAs) in axenic (27.75 ± 0.62%) and co-cultured (28.46 ± 0.28%)
I. galbana were similar. The most abundant MUFA in both axenic and co-
cultured I. galbana was elaidic acid (C18:1n-9 t). The specific MUFA
obtained from mono-cultured Marinobacter sp. was myristoleic acid
(C14:1n-5). Besides, the content of heptadecenoic acid (C17:1n-7) was
Y.-Y. Wang et al. Aquaculture 556 (2022) 738248

Fig. 2. Chlorophyll a content (A), Fv/Fm (B), protein content (C) and superoxide dismutase (SOD) activity (D) for Isochrysis galbana co-culture and mono-culture
during 14 d exposure time. Significance of the differences between mean values was determined with Student’s t-test. All the experiments were carried out in
triplicate with standard error as error bar, while asterisks (*) indicate significant difference at p value <0.05.

Table 1 Table 2
Saturated fatty acid composition (as a percentage of total fatty acids) of Iso­ Monounsaturated fatty acid composition (as a percentage of total fatty acids) of
chrysis galbana co-cultured with Marinobacter sp. for 14 days. Isochrysis galbana co-cultured with Marinobacter sp. for 14 days.
Saturated fatty Axenic I. galbana Axenic I. galbana Marinobacter sp. Monounsaturated Axenic I. galbana Axenic I. galbana Marinobacter sp.
acids (SFA’s) + Marinobacter sp. fatty acids (MUFA’s) + Marinobacter sp.

C12:0 ND ND 22.81 ± 0.66 C14:1n-5 ND ND 0.49 ± 0.02

C13:0 ND ND 1.10 ± 0.08 C16:1n-7 4.22 ± 0.16 5.15 ± 0.06* 7.47 ± 1.50
C14:0 8.9 ± 0.13 10.68 ± 0.07* 1.36 ± 0.16 C17:1n-7 1.13 ± 0.06 0.75 ± 0.01* 1.01 ± 0.18
C15:0 1.29 ± 0.09 0.87 ± 0.00* 0.32 ± 0.02 C18:1n-9 t 19.02 ± 0.22 18.68 ± 0.16 14.80 ± 0.22
C16:0 15.09 ± 0.37 14.55 ± 0.05 29.91 ± 1.03 C18:1n-9c 2.29 ± 0.09 2.62 ± 0.03* 3.55 ± 0.75
C18:0 2.24 ± 0.26 1.14 ± 0.20* 4.36 ± 0.30 C22:1n-9 1.09 ± 0.09 1.26 ± 0.02 ND
C20:0 0.73 ± 0.37 0.24 ± 0.00 ND ΣMUFA 27.75 ± 0.62 28.46 ± 0.28 27.32 ± 2.67
C22:0 2.16 ± 0.10 1.20 ± 0.02* ND
ΣSFA 30.41 ± 1.32 28.68 ± 0.34 59.86 ± 2.25 Asterisks (*) indicate significant difference at p value <0.05. ND = not detected.

Asterisks (*) indicate significant difference at p value <0.05. ND = not detected.

significantly decreased in co-cultured I. galbana. The levels of total
polyunsaturated fatty acids (PUFAs) were not significantly changed in
axenic I. galbana (40.38 ± 1.78%) compared with the co-cultures (42.05

4
Y.-Y. Wang et al. Aquaculture 556 (2022) 738248

Table 3 Therefore, it is necessary to find strategies to improve its production. In

Polyunsaturated fatty acid composition (as a percentage of total fatty acids) of our study, we identified a growth-promoting bacterial strain Mar­
Isochrysis galbana co-cultured with Marinobacter sp. for 14 days. inobacter sp. and DHA production of I. galbana can be enhanced by this
Polyunsaturated fatty Axenic Axenic I. galbana+ Marinobacter bacterium. As we known, this was the first study to find a bacterium that
acids (PUFA’s) I. galbana Marinobacter sp. sp. can both enhance the algal biomass and DHA production in I. galbana.
C16:2n-6 0.89 ± 0.08 1.13 ± 0.03* ND Our results lay a foundation for studying the interaction between algae
C18:2n-6 5.21 ± 0.54 3.47 ± 0.01 ND and bacteria and help improve the yield and quality of bait-microalgae
C18:3n-3 6.90 ± 0.10 6.07 ± 0.06* ND in aquaculture.
C18:4n-3 10.99 ± 13.91 ± 0.05* ND
When we explored the effects of Marinobacter sp. on the growth of
0.41
C20:2n-6 0.74 ± 0.07 0.39 ± 0.07* ND I. galbana, we found that the growth-promoting role of Marinobacter sp.
C20:3n-3 0.38 ± 0.04 0.21 ± 0.02* ND to I. galbana depends on the population density of the bacterium. With
C20:4n-6 0.33 ± 0.06 0.17 ± 0.00 ND the increase of bacterial density, the growth-promoting effects became
C20:4n-3 0.41 ± 0.08 0.15 ± 0.00* ND more obvious (Fig. 1). It is crucial to note that in cultures with a high
C20:5n-3 1.13 ± 0.12 1.16 ± 0.02 ND
C22:5n-6 2.22 ± 0.14 1.96 ± 0.03 ND
density of bacterial cells, the availability of light and nutrients to
C22:6n-3 11.18 ± 13.43 ± 0.10* ND microalgae cells is lower, and the growth rates and yields of valuable
0.14 compounds, such as carotenoids and fatty acids, decrease (Fuentes et al.,
ΣPUFA 40.38 ± 42.05 ± 0.39 ND 2016). Possibly Marinobacter sp. cell density in our study was not suf­
1.78
ficiently high to affect the availability of light and nutrients. Our results
Asterisks (*) indicate significant difference at p value <0.05. ND = not detected. also suggested that an appropriate algae/bacteria ratio was a crucial
factor to achieve high I. galbana growth in the co-cultivation system.
Similarly, the growth-promoting effect of Alteromonas macleodii on
± 0.39%). Our study clearly suggested that PUFAs were absent in I. galbana also depends on the population density of the bacteria (Cao
Marinobacter sp. The contents of hexadecadienoic acid (C16:2n-6), et al., 2021).
stearidonic acid (C18:4n-3), and DHA (C22:6n-3) in the co-cultured Our results also indicated in the presence of Marinobacter sp., the
I. galbana were significantly increased compared with the axenic content of chlorophyll a was always significantly higher than that of
I. galbana. However, the contents of α-linolenic acid (C18:3n-3), eico­ axenic algal culture (Fig. 2). This finding was consistent with a previous
sadienoic acid (C20:2n-6), eicosatrienoic acid (C20:3n-3) and eicosate­ study that Marinobacter sp. has positive effects on the growth of seven
traenoic acid (C20:4n-3) were significantly decreased compared with microalgal species (Ling et al., 2020). Besides, the co-cultivation of
the axenic I. galbana. Rhizobium sp. and Ankistrodesmus sp. results in an increment of up to
30% in chlorophyll (Do Nascimento et al., 2013). Likewise, a similar
3.4. The effects of Marinobacter sp. on the expressions of genes involved study has shown that the co-immobilization of Chlorella vulgaris and
in DHA synthesis pathway Azospirillum brasilense leads to increments of up to 35% in chlorophyll a
(Gonzalez and Bashan, 2000). The increase in the content of chlorophyll
ASE2, d4FAD, d5FAD, d6FAD, and d8FAD are key genes involved in a was indeed a manifestation of the algal growth promoted by Mar­
the addition of double bonds and carbon atoms to the growing fatty inobacter sp.
chain in the DHA synthesis pathway (Fig. S3). Based on RT-qPCR Stresses usually lead to the accumulation of reactive oxygen species
analysis, the expressions of these five genes were all up-regulated in (ROS). ROS are extremely harmful when their production exceeds the
co-cultures compared with the mono-cultures on all three time points (2 buffering capacity of the antioxidant system (Wang et al., 2019). It is
d, 4 d and 14 d after co-cultivation) (Fig. 3). Among them, the expression well known that SOD plays a key role in the removal of ROS. We found
of d8FAD was mostly enhanced (fold change = 12.99) on 14 d. that the SOD activity of I. galbana was always lower compared with the
control group during the co-culture period (Fig. 2). Similarly, co-culture
4. Discussion with A. macleodii severely inhibits the SOD activity of I. galbana
compared with the control group during the entire study period, which
I. galbana, as a high-quality bait microalgae species, has been widely may be related to the ability of A. macleodii to produce extracellular
used in aquaculture (Tremblay et al., 2007). However, mass cultivation antioxidants (Cao et al., 2021). Whether Marinobacter sp. can produce
of I. galbana is often impaired by different environmental factors. antioxidants is worthy of further study.
When we explored the effects of Marinobacter sp. on the fatty acid
composition of I. galbana, it is interesting to find that the production of
DHA was significantly improved by Marinobacter sp. DHA is one kind of
important nutrient for aquatic animals. It was reported that higher
protein synthesis in microalgae results in elevated expressions of omega-
3/6 fatty acid biosynthetic genes, namely desaturase and elongase,
which subsequently synthesize long-chain PUFAs, such as EPA and DHA
(Jeyakumar et al., 2020). As higher protein synthesis was found in co-
culture, we conjectured that the high protein content might be one
reason for the increased content of DHA. Similarly, the proportion of
unsaturated fatty acids of C. vulgaris-Mesorhizobium sangaii co-culture
was significantly increased compared with the pure algae culture (Wei
et al., 2019). They explained that this enhancement in co-cultures might
be due to the metabolic regulation and nutrient exchange between
microalgae and bacteria.
To further explore the reason for the enhancement of DHA produc­
Fig. 3. Real-time quantitative PCR (RT-qPCR) analysis of the expression of
genes involved in DHA synthesis pathway (ASE2, d4FAD, d5FAD, d6FAD and tion, the effects of Marinobacter sp. on the expressions of genes involved
d8FAD) in co-culture and mono-culture on three time points (2 d, 4 d and 14 d). in DHA synthesis pathway were studied. Our study showed that the
RT-qPCR data represent the mean ± standard error of three independent ex­ expression of ASE2, d4FAD, d5FAD, d6FAD, and d8FAD in the DHA
periments, while asterisks (*) indicate significant difference at p value <0.05. synthesis pathway significantly up-regulated in co-cultures. Therefore,

5
Y.-Y. Wang et al.
the increased content of DHA in co-cultures might be attributed to that
Marinobacter sp. stimulated the expressions of genes involved in the
DHA synthesis pathway. Phosphorus, as a major growth-limiting
nutrient for microalgal growth, is necessary for synthesizing vital bio­
molecules, such as DNA, RNA, and phospholipids (including DHA) (Liu
et al., 2017). Besides, it is interesting to find that Marinobacter sp. has the
ability to decompose and mineralize organic phosphorus. Therefore, we
proposed another hypothesis that Marinobacter sp. decomposed and
mineralized organic phosphorus by the alkaline phosphatase enzyme to
inorganic phosphorus that could be taken up by I. galbana. The increased
phosphorus intake might be the reason for the increased DHA synthesis.
However, additional studies are required to validate this hypothesis.
Marinobacter sp. exists in a wide range of environments, playing an
important in the biochemical cycles of the earth, and it can use a variety
of carbon sources to interact with metals (Singer et al., 2011). It has been
reported that Marinobacter sp. can promote the growth of dinoflagellates
and coccolithophores (Amin et al., 2009b). This Marinobacter strain is
capable of producing vibrioferrin (VF), an unusual lower-affinity dici­
trate siderophore. With the help of VF, the bacteria promote algal
assimilation of iron by facilitating photochemical redox cycling of iron
(Amin et al., 2009a). However, it has been reported that the VF-
producing strains are a small number of strains in the genus Mar­
inobacter, and most strains do not produce VF (Amin et al., 2007; Vraspir
and Butler, 2009). It was worth mentioning that we attempted to
determine the ability to produce VF of this bacterial strain. However, we
did not detect the production of VF in Marinobacter sp. (data not shown).
We also attempted to amplify the VF biosynthetic genes, while we didn’t
obtain the expected PCR product. Therefore, the mechanism for Mar­
inobacter sp. to promote the growth of I. galbana was not dependent on
the VF produced by Marinobacter sp. as reported in dinoflagellates and
coccolithophores. It is well studied that bacteria can secrete hormones,
e.g. indole-3-acetic acid (IAA), that stimulate the growth and meta­
bolism of microalgae (Amin et al., 2015; De Bashan et al., 2008). Our
study showed the potential of Marinobacter sp. to release IAA which
might have a significant positive impact on algal growth (Fig. S4).
Whether Marinobacter sp. enhances the growth of I. galbana by providing
it with IAA merits further study. Taken together, we are sure that the
metabolic regulation and nutrient exchange happened between Mar­
inobacter sp. and I. galbana. What kind of metabolic regulation and what
substances are exchanged merits further investigations.
Collectively, co-cultures of I. galbana and Marinobacter sp. altered the
metabolism and fatty acid contents of microalgal cells and increased the
levels of DHA. Therefore, such an approach could be applied in high-
value compound production and mariculture of microalgae.
5. Conclusion
In our present study, we selected a bacterial strain with a strong
ability to promote microalgal growth and DHA production in I. galbana.
Co-cultures of I. galbana and Marinobacter sp. altered the growth, the
content of chlorophyll a, Fv/Fm, soluble protein content, and SOD ac­
tivity of I. galbana. Besides, the presence of Marinobacter sp. promoted
the production of DHA and up-regulated the expressions of genes
involved in DHA synthesis pathway. Taken together, our current find­
ings provided valuable insights into the interaction between algae and
bacteria and helped improve the yield and quality of microalgae.
CRediT authorship contribution statement
Ying-Ying Wang: Investigation, Data curation, Formal analysis,
Writing – original draft, Writing – review & editing. Si-Min Xu: Data
curation, Writing – review & editing, Validation, Visualization. Jia-Yi
Cao: Funding acquisition, Conceptualization, Supervision, Writing –
original draft, Writing – review & editing. Min-Nan Wu: Data curation,
Writing – review & editing. Jing-Hao Lin: Validation, Visualization.
Cheng-Xu Zhou: Funding acquisition, Conceptualization. Lin Zhang:
6
Aquaculture 556 (2022) 738248
Funding acquisition, Writing – review & editing. Hai-Bo Zhou: Writing
– review & editing. Yan-Rong Li: Writing – review & editing. Ji-Lin Xu:
Funding acquisition, Conceptualization, Supervision. Xiao-Jun Yan:
Conceptualization.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Acknowledgements
This research was supported by the National Key Research and
Development Program of China (2018YFD0900702; 2018YFA0903003;
2018YFD0901504; 2019YFD0900400), Ningbo Science and Technology
Research Projects, China (2019B10006; 2019C10023), Zhejiang Major
Science Project, China (2019C02057), China Agriculture Research Sys­
tem of MOF and MARA, the National Natural Science Foundation of
China (31801724), Natural Science Foundation of Ningbo, China
(2019A610416), the Zhejiang Provincial Natural Science Foundation of
China (LY22C190001) and the Natural Science Foundation of Ningbo
Government (2021 J114).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2022.738248.
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Another random document with
no related content on Scribd:
 Some of the miners, instead of moving on to new
scenes of action when the gold began to give out,
have stayed on with their families, working a few
acres of land and occasionally panning out a little
gold.
 Much of the Yukon is unexplored, and bridges and
ferries are few, so the hunter and the prospector must
ford the rushing streams and make their own trails
through the country.
Indeed, many books might be made about the ups and downs of
the Klondike in the height of the gold fever. Men came here beggars
and went away millionaires, and millionaires lost fortunes and
became tramps. Gold was shipped out by the ton, and in the city of
Dawson it was spent by the pound. At the start, the town was what in
slang phrase is known as “wide open.” The scores of gambling
houses, saloons, and dance halls all made money. In one dance hall
twelve women were employed at $50 a week, besides the twenty-
five per cent. commission they received on the drinks and cigars sold
through their blandishments. One girl stated that her bar commission
for the first week amounted to $750. Another saloon had six beauties
to dance at $150 a week, and in many of the halls the women were
paid a dollar for a dance of five minutes.
I have before me a copy of a bill of fare of one of the old
restaurants. A bowl of soup cost $1 and a bowl of mush and milk
$1.25. A dish of canned tomatoes cost $2, a slice of pie 75 cents,
and a sandwich with coffee, $1.25. Beans, coffee, and bread were
$2, a plain steak was $3.50, and a porterhouse was $5.
A leading restaurant, which had a seating capacity of thirty-two,
employed three cooks, one of whom received $100 a week, and the
others $1 an hour. The waitresses got $100 a month. The restaurant
occupied a tent twenty by forty feet, which rented for $900 a month.
Carpenters were drawing $15 a day, and common labourers $10.
Skilled woodworkers got $17 a day, and journeymen tailors $1.50 an
hour. The ordinary charge for a sack suit was $125. Barbers made
from $15 to $40 a day, each receiving sixty-five per cent. of the
receipts of his chair. Four barber shops were in operation, and their
prices were $1 a shave, $1.50 for a hair cut, and $2.50 for a bath.
During that winter newspapers brought in over the trail sold for
$2 apiece. A weekly newspaper was started, known as the Yukon
Midnight Sun, which cost $15 a year, and a little later the Klondike
Nugget was issued weekly at 50 cents a copy.
Banks were soon established and did a big business in buying
gold dust and putting their notes into circulation. The first eight days
after it opened its doors, the Canadian Bank of Commerce bought
one and one half million dollars’ worth of gold dust. Some years ago
the old building in which that bank had its offices was burned, and
one of the clerks asked permission to work over the ground as a gold
claim. He wanted to recover the waste from the assay offices and
also the dust that had fallen on the floor from time to time in the
purchase of gold. His request was granted and his idea proved worth
thousands of dollars.
 CHAPTER XXXVII
A DREDGE KING OF THE KLONDIKE

Since I have come to western Canada I have acquired a

contempt for Aladdin. At every step here I am meeting common,
everyday men who are enslaving genii a million times mightier than
those of the Arabian Nights. They rub their magic lamps and
mechanical wonders spring up almost in a night. They give an order
and change the course of a river. They lift a hand and valleys are
turned upside down. Of all these conquerors of Nature in the
Klondike none has come up to Joseph W. Boyle, the famous dredge
king, who was once the most striking figure in this land of gold.
Joe Boyle started at the bottom and won great wealth and a
dominant position. In manner and thought he was as plain as a pipe
stem. A giant of a man, over six feet in his stockings, he was straight
and well formed. He had a big head, a broad, high forehead, and
eyes like blue steel. Yet he was a good companion and hail-fellow-
well-met with those he liked. He was a friend to his employees and
addressed them by their first names. They referred to him always as
“Joe Boyle” or “J. W. B.,” but they understood that he was the boss
and that everything must be done just as he said.
Boyle began his fight with life as a boy and kept it up until he
died after the World War. His father, who was a farmer living at
Woodstock, in eastern Canada, had planned that Joe should
become a lawyer or a preacher and with that end in mind had sent
him to college. This was too tame for “J. W. B.” He left school and
shipped before the mast as a sailor. Once, in going from the Island of
St. Helena around the Cape of Good Hope, his ship sprang a leak.
Boyle took charge of the crew at the pumps and kept them at work
for four thousand miles until they sailed into Bombay. When he had
risen to the position of quartermaster of a British vessel he gave up
the sea and came home.
A little later he struck out for the West, where he became trainer
and manager for Frank Slavin, the bare-knuckle champion prize
fighter. The two staked their all on Slavin’s success in a big fight,
which was lost. They had exactly fifty cents between them when they
decided to go up to the new gold mines of the Yukon. They “mushed”
it from Dyea over the mountains, and got to the Klondike shortly after
gold was discovered. For a time they worked together, and then
Boyle engaged in placer mining with Swift-Water Bill Gates.
At one time he and five or six companions ran out of supplies.
They had started for the “outside” through Chilkoot Pass, where a
blizzard caught them. Swift-Water was overcome, and Boyle carried
him back into camp on his shoulders. After that the party came to a
stream that only Boyle had the strength to cross. He took over the
others one at a time and they went on their way. When at last they
reached San Francisco they were given a big banquet and on the
menu cards was printed the story of what Boyle had done.
At this time Boyle was not doing as well as he had hoped at his
mining. He looked over the ground of the Klondike Valley and
conceived the idea that there was a fortune to be made in the earth
the miners had left. Boyle stood on a little hill above the Klondike
River, and determined to lease all the land within sight. This was
when the mining in the creeks was at its height and the valley was so
lean it was thought worthless.
Joe Boyle also staked a timber claim ten miles in length and
extending through and beyond the area of his mining claim.
Everyone laughed at his mining proposition, but he had to fight for
his timber. As soon as news of his application got out his competitors
at Dawson saw the authorities and had them require him to stake out
the whole ten miles of his claim. This stipulation was made at three
o’clock on the afternoon before the last day in which the title could
be perfected. Boyle started on foot that afternoon and tramped all
night, wading through swamps, blazing trees, and driving stakes to
define limits. The work was exhausting, but he kept on until he
thought he had marked out not less than fifteen miles. He got back to
Dawson at nine o’clock the next morning, only to find a number of
men ready to jump his claim if it had not been staked. When the area
was measured according to law, it was found that his stakes fell
short only twenty feet of the ten miles allotted. Boyle put in saw-mills
and made money out of his lumber and wood. He got from this same
claim the timbers needed in his gold dredging.
His lumber profits gave Boyle the money he needed to approach
capitalists about financing his mining concessions. He first formed an
alliance with the Rothschilds, by which he was to have one third and
they two thirds of the stock. The understanding was that they were to
furnish the money, amounting to some millions, and that Boyle was
to manage the property and superintend its development.
Then the Rothschilds tried to squeeze out “J. W. B.” They
questioned his title and planned a reorganization. Boyle carried the
matter to Ottawa; he fought them in the courts, where he got a
judgment in his favour for more than six hundred thousand dollars.
The Rothschilds then offered him a million dollars for his share of the
stock. He refused and in return made them an offer of four hundred
thousand dollars for the two thirds they held. At first they laughed,
but they finally reconsidered and accepted his proposition. Boyle
then formed another corporation, the Canadian Klondike Mining
Company, by which name the property is known to this day.
This company owns leases from the government of Canada that
give it the right to work the lower valley of the Klondike up to the
crest of the mountains on both sides of the river. The greater part of
its holdings lie in the wide bed between the hills through which runs
the swift-flowing river. At a distance it looks like farm land and when
the concessions were granted much of it was covered with gardens.
It had been cleared of woods by the first miners, who, it was
generally believed, had stripped the soil of its gold.
Joe Boyle thought otherwise. He reasoned, “If so much gold has
come from the valley there must be quantities of gold dust and
grains in the bed-rock underneath.” Working upon that supposition,
he became a rich man by handling gold-bearing earth carrying
values of only about twenty-six cents to the ton.
And this brings me to another of the wonders of engineering in
the Far North. It is a device invented by Boyle for keeping the hydro-
electric plant running throughout the winter, notwithstanding the fact
that the temperature at times falls to seventy degrees below zero.
That is so cold that if you should attempt to run a sprayer such as is
used in an orchard the water would turn to ice before it fell to the
ground. At such times some of the streams have seven feet of ice
over them and many are solid. Nevertheless, Boyle turned a branch
of the Klondike River into a ditch six miles long and dropped it down
upon turbines with a fall which he said would generate electricity to
the amount of ten thousand horse-power a day all the year through.
Joe Boyle knew that the waters of the Yukon and the Klondike
flow under the ice all winter long and that there is an air space
between the water and the ice overhead. He concluded that, on the
principle of the double walls of an ice house or a thermos bottle, it
was this dead air space that kept the running water from freezing.
The only thing necessary was to make Nature furnish the thermos
bottle. This Boyle did. He filled his ditch to the top and allowed a
sheet of ice to freeze a foot or so thick upon it. He then lowered the
level of the water two feet, leaving a running stream four feet deep,
with an air space above. He next installed electric heaters
underneath to help keep the water from freezing. In this way he
made the water warm itself, for the stream thus kept moving
generated the electricity for the heaters, each of which required
current equal to one hundred horse-power.
I went out yesterday in an automobile to North Fork, thirty miles
up the Klondike Valley, to see this electric plant. The ditch is thirty
feet wide, about six feet in depth, and six miles long. The water
drops down through great pipes, with a fall of two hundred and
twenty feet on the turbines. I asked one of the men how Mr. Boyle
got the idea of electrically heating the water and was told it came to
him one morning at breakfast. The family had toast and eggs, and
were browning the bread on the electric toaster. As he looked at it,
Boyle thought that he might employ the same principle in keeping
the water from freezing. His men made out of telephone wire a
gigantic toaster somewhat like a woven-wire bed spring. This was
properly insulated, dropped into the ditch, and connected with the
electric plant.
In 1914 Boyle was forty-seven years old and in the prime of his
vigour. Moreover, he had just won a million dollars in a suit against
the Guggenheims and so had plenty of cash for any adventure. He
organized a machine gun battery of fifty gunners, picked men of the
Yukon, and offered them to the Allied armies. To his great distress,
his battery was broken up and scattered through the forces. He went
to London and from there was sent into Russia to help in keeping
transportation open. On one occasion he reported to the chairman of
the Soldiers’ Committee, who was inclined to be nasty.
“Were you sent here because you were the best man they could
find on the Western Front?” he demanded of Boyle.
“Possibly so,” was the reply. “And now, you answer me this. Are
you the best man on your committee?”
“I am,” answered the chairman, expanding his chest.
“Very well, I will meet you man to man,” said Boyle, as he
unbuttoned his coat and doubled his fists. He had no more trouble
with that chairman.
 Starting with a capital of fifty cents, Joe Boyle
made a fortune by gleaning gold from abandoned
workings. Then he gave up mining to go to war and
became almost as famous in Eastern Europe as in
the Klondike.
 To get the water for washing down the gold-
bearing gravel of the Klondike hills, millions of dollars
were spent in building ditches, flumes, and pipes from
the Tombstone Mountains, seventy miles away.
When Russia gave up, Colonel Boyle went over into Rumania,
where he became a national hero. He undertook all sorts of
dangerous and important missions. For instance, when the
Bolsheviki were beginning to get the upper hand, he offered to go to
Moscow to bring back the national treasure of Rumania, which had
been sent there for safe keeping. He got into Moscow, loaded
millions of dollars’ worth of bank notes and securities on a special
train, and started back. On the way the engineer of the train
deserted, leaving his boilers without water or fuel. Boyle and his
helpers carried water in buckets from the nearest station and cut
wood for the fire. Though he had never driven a locomotive before,
Boyle climbed into the cab and got the train and its treasure across
the border. Later he turned the Russian Black Sea fleet pro-Ally,
arranged peace terms between Rumania and the Bolsheviki, and
saved sixty Rumanian deputies from banishment to Sebastopol.
After the Armistice he was commissioned to superintend the
distribution of the food and supplies bought for the country with the
Canadian credit of twenty-five million dollars. Then he became
interested, with the Royal Dutch Shell Transport Company, in oil
concessions in Caucasia.
In the course of his many adventures in Rumania, Colonel Boyle
flew so high and so fast in airplanes that he suffered a sort of
paralytic stroke. During his illness he was attended for two months
by Queen Marie and her daughter, who did everything they could to
show their appreciation of his service to their country. He finally
recovered, but when in England on his way back to Canada, he died
of heart failure.
 CHAPTER XXXVIII
THE ROYAL CANADIAN MOUNTED POLICE

Everyone has heard of the Royal Canadian Mounted Police.

They constitute one of the most remarkable military forces in
existence, with an amazing record for the capture and punishment of
criminals in the frontier lands of the Dominion. I have met with the
Mounted Police in all parts of Canada, have visited the headquarters
in Ottawa and the training station at Regina, and have talked here at
Dawson with the inspector in charge of the Yukon division. I find the
service a gold mine of stories, and fully deserving its reputation for
maintaining law and order on the fringes of civilization.
Our own “wild and woolly West” has disappeared, but Canada
still has vast areas of undeveloped country into which white men are
pushing their way under conditions similar to those in the United
States a generation or two ago. But where our frontier was notorious
for its lawlessness, that of the Dominion is equally noted for its few
crimes. In the Canadian Northwest a “bad man” cannot long escape
the strong arm of the law, and in nine cases out of ten he meets with
punishment both swift and sure.
From the wheat lands adjoining our border to the gold rivers of
the Yukon, from the Great Lakes to the Arctic Ocean, the settler, the
prospector, or the trader can lie down to sleep at night with little fear
for his safety. That this is so is chiefly due to this police force.
Detachments of the Royal Canadian Mounted Police are now
located all over Canada. They are to be found in the thickly
populated centres as well as in the Far North. But it was as a frontier
police that the organization was first created, and it was in the
Northwest Territories that its reputation was made. It has its stations
about Hudson Bay, along the Peace River, on the banks of the
Mackenzie, and on the shores of the Arctic Ocean. The latest posts
established are those on the north coast of Baffin Island, opposite
Greenland, and on Ellesmere Island, less than one thousand miles
from the North Pole.
The duties of the Mounted Police are widely varied. They are
especially charged with the enforcement of federal statutes, and are
wholly responsible for law and order in the Northwest Territory, the
Yukon, the national parks, and the Indian reservations. Elsewhere
the organization coöperates with provincial authorities and the
federal departments. It looks after such matters as violations of the
customs, of excise regulations, the circulation of radical or
revolutionary propaganda, the improper storing of explosives, and
the debauching of the Indians. Special patrols are sometimes sent
out to strengthen the hands of the Indian Department when unrest is
reported among their charges. Some are detailed to see that the
betting at the race tracks in the various provinces does not infringe
upon the laws, and others to escort trainloads of harvest workers to
their destinations and prevent disorders on the way. Patrols go for
hundreds of miles by dog sled into the Far North to keep order and
investigate crimes among the Eskimos.
The actual discharge of these duties leads to a variety of
activities. The Mounted Police patrol the United States border to
guard against smuggling of liquor, Chinese, and narcotics. They ride
about the newly colonized districts, visiting the homes of the settlers
and watching for cattle thieves. Any complaint of disorder or law
breaking is promptly investigated, and a member of the force may
spend months in the rôle of detective, seeking evidence or making a
search for a suspected man.
The Mounted Police have cut many of the trails of the Far North.
When the big gold strikes were made in the Klondike, they built the
first road through the wilds of the Yukon, and they have opened up
parts of the Canadian Rockies to prospectors. Whenever a new gold
district is discovered, or an oil find is reported, the Mounted Police
are among the first on the scene, and every one knows that the law
is at hand. That is why the Klondike was peaceable during gold rush
days, while in Alaska, across the international boundary, notorious
“bad men,” such as “Soapy Smith” and his gang, held almost
undisputed sway for a time.
The Mounted Police sometimes erect shelters along the new
trails, in which they place stores of food for use of prospectors in an
emergency. They often bring relief to those in the wilds rendered
helpless through injury, disease, or insanity. They settle on the spot
minor disputes, especially among the Indians and Eskimos,
sometimes perform marriages, and, as the Dawson inspector said to
me to-day, do about everything any occasion may require except
grant divorces. In extreme cases, a member of the force may arrest
his man, try his case, sentence him to death, and, finally, act as
clergyman, executioner, and coroner. It is the almost inviolate rule of
the organization, however, that a prisoner must be brought in alive
and given his chance at a fair trial.
 “Bring in your man” is the law, stronger than any
legislative enactment, of the Mounted Police. The
reputation established by this unique force for never
giving up is one of the reasons for its astonishing
success.
 With the increase of crime, especially murder,
among the Eskimos of the Far North, the Mounted
Police now have established several stations in the
Arctic, including one on Ellesmere Island, in the Polar
Sea.
 All these activities are carried on by a body of only a little more
than a thousand men, scattered from the Maritime Provinces to the
Alaska boundary. Here in the Yukon there are but fifty-one men, for
whom horses and dogs furnish a part of the transportation.
To get into the service a man must have a good character, a
sound body, and some education. Most of the men speak both
French and English. Recruits must be between the ages of twenty-
two and forty, unmarried, and expert horsemen. The term of
enlistment is three years, with reënlistments permitted. Many of the
present force have been long in the service. In their training at
Regina, much attention is paid to shooting with both rifle and pistol,
and in the latter the Mounted Police now hold the championship of all
Canada. Many of them are young Englishmen who have failed to
make their fortunes and some are younger sons of the nobility. In the
old days a son of Charles Dickens, the novelist, served beside a
former circus clown and the brother of a baronet.
The inspector of this body at Dawson is the military ruler of a
region bigger than Germany. It begins at the south, within thirty miles
of the Pacific Ocean, and extends northward to Herschel Island, near
where the Mackenzie River flows into the Arctic. It is about a
thousand miles long and several hundred miles wide. The inspector
tells me that his force is scattered all over this territory, from White
Horse, at the end of the White Pass Railway, to Rampart House, on
the Arctic Circle. When I asked him about the work of his force, he
said:
“Each of our constables has one or two men with him, and
sometimes an Indian or so. Together they patrol the whole country.
They make long trips to the mines, and report what is going on
among the prospectors. In out-of-the-way places they keep order
among the Indians and the Eskimos. They also look after the poor
and the insane. Recently we heard that a dangerous lunatic was at
large over in the Donjek District. Our patrol went after him and
brought him several hundred miles through the country to White
Horse, whence he was later sent to an asylum. Last year our men
penetrated to regions never visited before; they frequently make trips
of hundreds of miles by dog sled.”
 “But how can you keep track of the people in such a large
territory?” I asked. “Your whole land is a wilderness, and for more
than half the year it is all snow and ice.”
“Each hotel and road house is required to keep a daily record of
all who stop there,” he replied, “and I may say that we know about
where every man in the territory sleeps every night. We are informed
of all the passengers who start up or down river, and get reports from
every telegraph station they pass on the trip. When a steamer leaves
White Horse for Dawson the purser hands in the names of his
passengers and they are telegraphed here. If any one gets off on the
way his name is wired to us, and we check up the list when the boat
comes in. If three men set off in a canoe, the report on that canoe as
it passes the next telegraph station will show us if one of them is
missing. The patrols also send in reports of the names and business
of all newcomers in their districts.”
“Give me some idea of the amount of crime committed in your
territory.”
“Our record is fairly good,” replied the inspector of the Mounted
Police. “Last year we investigated forty-two cases, only eight of
which were under the criminal code. Out of this total of forty-two, we
secured thirty-seven convictions. Remember that this is for an area
as big as France, and for a population made up largely of
frontiersmen, miners, Indians, and Eskimos. Most of the time we
have so few bad characters in jail here that it is difficult to keep our
barracks in order and the lawn properly mowed. Just now we have
two women serving terms for picking the pockets of men who were
drunk. They work in the jail laundry, so we are sure of help in our
washing for the rest of the year.
“We have had but few murders in our territory,” the inspector
continued. “The average was less than one a year for the first twenty
years after the big rush to the Klondike, and in every case, without
exception, the guilty were caught and executed. There are some
interesting stories connected with crimes in this part of the world.
Take, for instance, one that occurred in Alaska. The murdered man
was a miner who had been killed by an Indian at the close of the
season when the miners were about to leave for the winter. They
had not time to follow the Indian, but they went to the chief of his
tribe and told him that he must catch the murderer and have him
ready for them when they returned in the spring. When the spring
came they went to the chief and demanded the man. He replied:
“‘Me got him all right. You come see.’ He thereupon took them to
the back of the camp and showed them a dead Indian frozen in a
large block of ice. As they looked, the chief continued:
“‘We got him last fall. We know you kill him in spring, so we
shoot him in fall. What use feed him all winter?’
“We had a case of a miner who inveigled two young men with
money to go with him in a canoe two hundred miles down the Yukon.
From there they were to make their way inland to a gold prospect the
miner had located. As they camped, the miner had one of the men
build a fire, while he took the other off to hunt game. Within a short
time the man at the camp heard a shot and later the miner came in
and said they had killed a bear about a mile away and wanted the
man at the camp to go with him to bring in the meat. The two started
off together, the miner walking behind. The stranger began to think
that all was not right. He turned his head quickly and found that his
companion had raised his rifle and was drawing a bead on him. He
grappled with him and succeeded in getting the gun. He ran away
and finally got to Dawson, where he notified us. We watched the
river and within a few days the old miner came down in a boat. Our
men arrested him and then went back to the camp and found the
body of the man who started out to hunt bear. The murderer was
tried in a month and hanged two months later.”
“Do you ever have any lynchings?” I asked.
“I do not believe there has ever been a lynching in all Canada,”
said the inspector. “Certainly I never have heard of one in the Yukon.
Neither do we have hold-ups such as are not uncommon, I am told,
in the United States.”
The inspector’s reference to hold-ups reminded me of a story of
a highwayman I heard at the Mounted Police headquarters in
Ottawa. A road agent held up a man and a woman who were riding
through the hills. He covered them with his revolver and made the
man dismount so he could go through his pockets. The woman was
sitting on her horse, congratulating herself upon her escape, when
the robber stepped up to her, saying, “Beg pardon. Just a moment,
madam.” He thereupon gently raised her skirt to her knees, thrust his
hand into her stocking, and took out her money. He seemed to know
just where it was, and there was no waste effort.
“One of the classics of our service”—it is the inspector who is
speaking once more—“is the King-Hayward case. Edward Hayward,
a young Englishman, was killed in the wilds around Lesser Slave
Lake. He had gone up there from Edmonton to hunt with Charles
King, an American from Salt Lake City. Some weeks later an Indian
notified one of our sergeants that two men had come into the country
and one of them had disappeared. The officer got on the trail, went
to the last camp fire, where the Indian reported seeing both men,
and sifted the ashes. He found three hard lumps of flesh and a bit of
skull bone. Near the camp fire was a little pond. In this Indian women
were set to work to fish up with their toes any hard substance they
might find in the ooze. They brought up a stick-pin of unusual design
and a pocketbook. The pond was drained and on the bottom was a
shoe with a broken needle sticking in it. The sergeant then examined
the ashes of the fire with a microscope, which revealed the eye of
the broken needle.
“King was tracked down and arrested, and Hayward’s brother
was brought on from England to identify the trinkets of the murdered
man. It took the sergeant eleven months to complete his case, and
he had to bring forty Indian and half-breed witnesses from Lesser
Slave Lake to Edmonton to testify at the trial. But King was finally
convicted and hanged. All this cost the Canadian government more
than thirty thousand dollars, yet it was not considered a waste of
money.”
I inquired of the inspector the cause of most of the crime in his
division. He replied: