Dietary Methionine Hydroxy Analogue Supplementation Benefits On Growth Intestinal Antioxidant Status and Microbiota in Juvenile Largemouth Bass Micropterus Salmoides Ye Zhao Full Chapter PDF
Dietary Methionine Hydroxy Analogue Supplementation Benefits On Growth Intestinal Antioxidant Status and Microbiota in Juvenile Largemouth Bass Micropterus Salmoides Ye Zhao Full Chapter PDF
Dietary Methionine Hydroxy Analogue Supplementation Benefits On Growth Intestinal Antioxidant Status and Microbiota in Juvenile Largemouth Bass Micropterus Salmoides Ye Zhao Full Chapter PDF
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Aquaculture 556 (2022) 738279
Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture
A R T I C L E I N F O A B S T R A C T
Keywords: To study the possible impacts of dietary methionine hydroxy analogue (MHA) on growth, intestinal antioxidant
Methionine hydroxy analogue status, and intestinal microbiota in juvenile largemouth bass (LMB) Micropterus salmoides (14.49–14.58 g), we
Growth conducted a feeding trial for 84-days. Four hundred and eighty fish were randomized into four dietary MHA
Antioxidant
treatments (0.0, 3.0, 6.0, and 9.0 g/kg diet). Results revealed that dietary MHA supplementation: (1) dramati
Intestinal microbiota
cally improved growth of juvenile LMB; (2) markedly decreased intestinal protein and lipid peroxidation, and
increased intestinal antioxidant activities via Keap1/Nrf2 axis; (3) increased the richness and diversity of in
testinal microbial community; (4) changed intestinal microbiota composition, increased the abundances of
Proteobacteria, Bacteroidetes, and Actinobacteria, and decreased the abundances of Firmicutes and Cyanobacteria at
the phylum level; increased the abundances of Bifidobacterium and Bacillus and decreased the abundance of
Bacteroides at the genus level. Collectively, dietary MHA supplementation has potential benefits in growth, in
testinal antioxidant capacity, and intestinal microbiota in LMB. The dietary optimal Met level of LMB
(14.49–131.06 g) was recommended to be 14.49 g/kg diet, corresponding to 8.30 g/kg diet sourcing from basic
diet and 4.19 g Met/kg diet sourcing from MHA based on specific growth rate.
* Corresponding authors at: Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Ya’an 625014,
China.
E-mail addresses: [email protected] (X.-Q. Zhou), [email protected] (J. Jiang).
1
These three authors contributed equally to this project.
https://doi.org/10.1016/j.aquaculture.2022.738279
Received 3 June 2021; Received in revised form 18 April 2022; Accepted 19 April 2022
Available online 27 April 2022
0044-8486/© 2022 Published by Elsevier B.V.
Y. Zhao et al. Aquaculture 556 (2022) 738279
fish species (Pan et al., 2017; To et al., 2020; Keembiyehetty and Gatlin, isolipidic diets (crude protein, 42.0%; crude lipid, 12.5%) is listed in
1995). Whether MHA could be used as a source of Met to promote LMB Table 1. The MHA (purity 88%, Sumitomo-chemical, Tokyo, Japan) was
growth is no report, it is worthy to explore. added to the experimental diets at levels of 0.0 (control), 3.0, 6.0, and
Fish growth is associated with the integrity of intestine, which is 9.0 g/kg. The amount of MHA supplementation was compensated by
associated with its antioxidant status (Jiang et al., 2015). Reactive ox wheat flour. The Met concentrations in four experimental diets were 8.3,
ygen species (ROS) are generated during normal digestive and absorp 11.0, 13.6 and 16.3 g/kg of dry diet (Table 2). The feed materials were
tive progress (Zhang et al., 2013), which excessive production will result crushed and thoroughly mixed with soybean oil and water in a blender
in oxidative damage and disturb integrity and function in fish intestine and then the diets were prepared using a twin-screw extruder (MY-165)
(Jiang et al., 2010). Recent studies indicate Met in grass carp Cteno equipped with a 3-mm diameter mold. All diets were air-dried in a cool
pharyngodon idella (Wu et al., 2017) and rohu Labeo rohita (Noor et al., place, and stored at 4 ◦ C until used.
2021) and MHA in Jian carp Cyprinus carpio var. Jian (Feng et al., 2011)
and grass carp (Pan et al., 2017) improve intestinal antioxidant status 2.2. Fish management and feeding
and depress lipid and protein oxidation via increasing antioxidant en
zymes activities and non-enzymatic antioxidant contents. The expres The feeding trial was performed at Ya’an Experiment Station
sion of the antioxidant enzyme genes is regulated by Keap1 (Kelch-like (Sichuan, China). Fish were provided by a local farm (Sichuan, China)
ECH-associated protein 1) - Nrf2 (nuclear factor erythroid 2-related and acclimated to the basal diet and culture conditions for 28 days.
factor 2) axis in fish (Giuliani and Regoli, 2014). The latest data Before the feeding experiment, all the fish were fasted for 24 h. The 480
shows that Met enhances antioxidant capacity in the kidney and liver of juvenile LMB with a similar size (14.49–14.58 g) were randomized into
blunt snout bream Megalobrama amblycephala by regulating Nrf2 and 12 concrete tanks (200 × 100 × 105 cm3) with 40 fish each replicate.
Keap1 mRNA expressions (Ji et al., 2020). Dietary Met also improves Twelve experimental tanks were randomly distributed into four groups,
hepatic antioxidant capability of large yellow croaker Larimichthys cro each group corresponding to one experimental diet. The fish were hand-
cea fed with high lipids diets (Li et al., 2021). In addition, study in grass fed for 84 days under natural photoperiod, twice a day (8:00 am and
carp indicates that dietary MHA improves intestinal antioxidant status 18:00 pm) until apparent satiety. The uneaten diets were collected thirty
by modulating Keap1-Nrf2 axis to up-regulate antioxidant enzyme genes minutes after the feeding, then oven-dried at 65 ◦ C to determine feed
mRNA levels, which contributes to alleviate oxidative damage (Pan intake (FI). The water quality parameters were measured as follows:
et al., 2017). Dietary supplementation with MHA improves the hepatic water temperature ranged from 23 to 27 ◦ C, water pH was 7.0 to 7.5, and
antioxidative capacity in turbot Scophthalmus maximus (Hu et al., 2015). the dissolved oxygen content was about 6.0 mg/L.
However, whether MHA can improve the antioxidant status by regu
lating Keap1-Nrf2 axis in LMB are poorly known. 2.3. Sample collection
The intestinal microbiota plays a vital role in optimal growth (Meng
et al., 2021; Tao et al., 2019; Zhao et al., 2014). Diet is an important The LMB from each tank were starved for 1 day, then weighted and
factor that affects the establishment and composition of intestinal counted. Twenty-one fish of every tank were randomly collected for
microbiota (Jang et al., 2019; Tan and Sun, 2020). Dietary Met increases sampling and anesthetized with benzocaine in dose of 50 mg/L. Three
intestinal Lactobacillus and Bacillus counts and decreases Escherichia coli fish per tank were randomly selected and kept at − 20 ◦ C to determine
and Aeromonas counts in Jian carp (Tang et al., 2009). Study in mice the whole-body composition. The intestines of eighteen fish from each
(Wallis et al., 2020) also shows that dietary Met restriction alters the
intestinal microbiome (Bacteroidaceae, Verrucoccaceae, and Rummino
Table 1
coccaceae). Dietary Met deficiency increases the proportion of beneficial Composition and nutrients content of diets (g/kg).
bacteria, and decreases the proportion of pathogenic bacteria in colonic
MHA levels 0.0 3.0 6.0 9.0
contents of mice (Yang et al., 2019). Meanwhile, dietary MHA in dairy
cows increases the abundance of Fibrobacter succinogenes and Rumino Fish meal 250.0 250.0 250.0 250.0
Soybean meal 220.0 220.0 220.0 220.0
coccus flavefaciens and depresses the abundance of bacteria that were
Corn protein meal 100.0 100.0 100.0 100.0
involved in trans-10 isomer production (Martin et al., 2013; Pitta et al., Gelatin 60.0 60.0 60.0 60.0
2020). A report in broilers indicates that MHA suppresses proliferation Soybean oil 100.0 100.0 100.0 100.0
of acid-producing bacteria including Roseburia and Collinsella (Wu et al., Wheat flour 165.0 162.0 159.0 156.0
2020). In swine, MHA inhibits ileal bacterial growth and metabolism CaH2PO4 40.0 40.0 40.0 40.0
MHA 0.0 3.0 6.0 9.0
effectively under in vitro conditions, but this effect was not detected in Vitamin premix1 10.0 10.0 10.0 10.0
vivo (Apajalahti et al., 2009). These results suggest that the effect Met Mineral premix2 20.0 20.0 20.0 20.0
and MHA on intestinal microbiota remains inconclusive under different Amino acid premix3 11.8 11.8 11.8 11.8
animal species and different experimental conditions. Whether they Choline chloride (50%) 10.0 10.0 10.0 10.0
Ethoxyquin (30%) 0.5 0.5 0.5 0.5
could be used as available modulators for intestinal microbial system
Microcrystalline cellulose 12.7 12.7 12.7 12.7
needs a further study. Moreover, a lack of research has been done about Nutrients content4
the impacts of dietary MHA on the intestinal microbiota in LMB, it is Crude protein 419.6 417.8 417.7 416.7
worthy to study. Crude lipid 124.6 126.8 127.0 125.2
That all, there is no study examines the effects of dietary MHA on the Crude ash 105.8 106.2 105.7 106.4
Moisture 96.2 94.1 93.7 94.9
intestinal antioxidant status and microbiota of LMB. Therefore, the
current work was undertaken to evaluate the influences of dietary MHA MAH = Methionine hydroxy analogue.
1
as Met source on growth, intestinal antioxidant status and intestinal Vitamin premix (IU or mg/kg diet): biosterol, 18,000 IU; vitamin D3 9000
microflora in LMB. The results might provide partial theoretical evi IU, 0.001; vitamin K3, 16.56; thiamine, 20.03; riboflavin, 54; vitamin B6, 33.21;
dence to the application of MHA in LMB. cyanocobalamin, 0.27; α-tocopherol, 28.00; ascorbic acid, 150.00; niacinamide,
26.00; calcium pantothenate, 25.00; folic acid, 1.00; biotin, 0.06; inositol,
400.00.
2. Materials and methods 2
Mineral premix (mg/kg diet): Cu, 5.00; Zn, 37.00; Mn, 7.00; Fe, 30.00; I,
1.10; Se, 0.25; Co, 0.20; Mg, 600.00.
2.1. Experimental design and diets 3
Amino acid premix (g/kg diet): His, 0.95; Ile, 1.97; Lys, 1.67; Val, 1.69; Cys,
2.08; Tyr, 2.88;Thr, 2.69.
The formulation and composition of four isonitrogenous and 4
Crude protein, crude fat and ash was measured by using the AOAC Methods.
2
Y. Zhao et al. Aquaculture 556 (2022) 738279
Table 2 Table 3
Amino acid composition of experimental diets (as DM-basis, g/kg). Primer sequences and OAT (◦ C) of genes selected for analysis by real-time PCR.
MHA levels 0.0 3.0 6.0 9.0 Name Sequences OAT Accession number
3
Y. Zhao et al. Aquaculture 556 (2022) 738279
quality base calls were removed, and the clean reads were obtained. (14.49–131.06 g) was recommended to be 12.49 g/kg diet based on
Sequences were clustered to the same operational taxonomic units SGR, corresponding to 8.30 g Met /kg diet sourcing from basic diet and
(OTUs) using Uparse (V7.0.1001) at ≥97% identity. The OTU repre 4.19 g Met/kg diet sourcing from MHA (Fig. 1).
sentation sequence was compared with the GreenGene database using Data on body composition of LMB fed different MHA diets are listed
RDP classifier (V2.2), and then annotate taxonomic information. The in Table 5. Dietary supplementation of MHA at 3.0, 6.0, and 9.0 g/kg
abundance information of OTUs were normalized using a standard dramatically enhanced whole-body protein, lipid content, PPV, and LPV
sequence number corresponding to the sample with the fewest se (P < 0.05). The LMB fed diets added with 3.0 and 6.0 g/kg MHA had
quences, which was used to alpha diversity analysis. Alpha diversity was markedly higher ash and APV than these fish fed control and 9.0 g MHA/
calculated by QIIME (V1.7.0) by the species richness estimator (Chao1 kg diet (P < 0.05). Dietary MHA had no significant effects on moisture
and ACE) and diversity indices (Shannon and Simpson). Rarefaction (P > 0.05).
analysis and Venn diagrams were performed using Mothur (V1.30.1)
and R program package. A principal coordinate analysis (PCoA) was
carried out by the WGCNA package and ggplot2 package in R software 3.2. Morphological index
(V2.15.3).
There was no significant difference in intestinal weight (IW) and
2.8. Statistical analysis intestosomatic index (ISI) among different MHA levels (Table 6, P >
0.05). The LMB fed diet with 3.0 g/kg MHA had higher intestinal length
The values were presented as means ± stand error (mean ± SEM). (IL) and relative gut length (RGL) than these fish fed the control diet
The experimental data were done by IBM SPSS Statistics 27.0 and P- (Table 6, P > 0.05).
value<0.05 was considered significant. Differences of the obtained data
among different dietary MHA treatment were determined using ANOVA
and Duncan’s multiple-range test. The Pearson correlation analysis and 3.3. Biochemical parameters in intestine
regression analysis were conducted. The quadratic regression model was
used to estimate the optimal MHA level for LMB. With daily feed con Results of the biochemical parameters in intestine of LMB are given
sumption (DFC) as a covariate, one-way ANCOVA was used to compare in Table 7. Fish in 3.0 g/kg diet group presented the lowest MDA and PC
final body weight (FBW). concentrations (P < 0.05). Meanwhile, the ASA, AHR, CAT, GPX, GST,
and GR activities of fish fed 3.0 g MHA/kg diet were markedly higher
3. Results than that of fish fed control, 6.0, and 9.0 g MHA/kg diets (P < 0.05). The
higher GSH content and T-SOD activity in intestine were recorded in fish
3.1. Growth parameters and body composition fed diet with 3.0, 6.0 and 9.0 g MHA/kg diets (P < 0.05). The expression
pattern of antioxidant-related genes in intestine of LMB are displayed in
Table 4 provides growth parameters of LMB fed four different diets Figs. 2 and 3. The SOD, CAT, and GCLC mRNA expressions were
for 84 days. Dietary MHA level did not affect the survival rate (SR). The remarkably upregulated in LMB fed 3.0 g MHA/kg diet (P < 0.05). The
higher growth parameters including final body weight (FBW), specific higher relative GPX, GST, and GR mRNA expressions were found in 3.0
growth rate (SGR), and feed intake (FI) were observed in 3 and 6 g/kg and 6.0 g/kg MHA groups compared to the control group (P < 0.05).
MHA levels in diet (P < 0.05). But daily feed consumption (DFC) was no Dietary MHA levels at 3.0, 6.0, and 9.0 g/kg dramatically enhanced Nrf2
significant difference (P > 0.05). The LMB receiving 3.0 and 6.0 g/kg gene expression. Besides, the lowest value of Keap1 gene expression was
dietary MHA reflected higher feed efficiency (FE) than these fish fed the recorded in fish fed the 3.0 g MHA/kg diet (P < 0.05).
control diet (P < 0.05). The dietary optimal Met level of juvenile LMB Fig. 4 displays nuclear Nrf2 and cytosolic Keap1 protein levels. Diets
containing 3.0, 6.0, and 9.0 g MHA/kg diet significantly increased nu
Table 4 clear Nrf2 protein level, while no statistical difference was observed
The IBW, FBW, SR, PWG, SGR, FI, and FE of LMB fed diets with different level of among other three diet groups. The lowest level of Keap1 protein was
MHA for 84 d1. detected in fish fed 3.0 g/kg MHA diet.
MHA (g/ 0.0 3.0 6.0 9.0
kg)
Y = -0.004X2 + 0.0999X + 1.96
SR2 100 ± 0.00 99.17 ± 1.44 100 ± 0.00 100 ± 0.00 R2 = 0.7563, P<0.05
IBW 14.54 ± 0.04 14.50 ± 0.01 14.55 ± 0.03 14.49 ± 0.02
119.11 ± 127.96 ± 123.88 ± 120.61 ±
FBW
0.82a 0.94c 0.74b 0.82a
3
SGR 2.48 ± 0.03a 2.62 ± 0.04c 2.55 ± 0.01b 2.50 ± 0.01a
6
DFC 2.02 ± 0.02 1.93 ± 0.06 1.97 ± 0.00 2.00 ± 0.02
LMB = largemouth bass; MAH = methionine hydroxy analogue; IBW = initial
body weight, FBW = final body weight with DFC as a covariate; SR = survival
rate; PWG = percent weight gain; SGR = specific growth rate; FI = food intake;
4
Y. Zhao et al. Aquaculture 556 (2022) 738279
Table 5 Table 7
Body composition (as wet-basis, %), PPV, LPV, and APV of LMB fed diets with The MDA, PC, and GSH contents (nmol mg-1 protein), ASA, AHR, T-SOD, CAT,
different level of MHA for 84 d1. GPx, GST, and GR activities (U mg-1 protein) in intestine of LMB fed diets with
MHA (g/kg) 0.0 3.0 6.0 9.0
different level of MHA for 84 d1.
MHA (g/ 0.0 3.0 6.0 9.0
Moisture 68.21 ± 0.59 67.76 ± 0.07 67.92 ± 0.43 68.23 ± 0.17
kg)
Protein 16.90 ± 0.24a 17.39 ± 0.18b 17.33 ± 0.23b 17.51 ± 0.01b
Lipid 8.51 ± 0.12a 8.82 ± 0.06b 9.05 ± 0.09b 9.81 ± 0.06c MDA 1.38 ± 0.01d 0.41 ± 0.02a 0.51 ± 0.01b 0.97 ± 0.02c
Ash 3.84 ± 0.02a 3.89 ± 0.05b 3.89 ± 0.02b 3.81 ± 0.01a PC 1.43 ± 0.03c 1.07 ± 0.01a 1.10 ± 0.01ab 1.14 ± 0.01b
PPV2 38.47 ± 0.33a 42.52 ± 0.63c 40.30 ± 0.08b 40.20 ± 0.03b 303.61 ± 348.39 ± 343.14 ±
ASA 376.12 ± 3.00c
LPV3 67.37 ± 0.27a 70.00 ± 1.24b 72.37 ± 0.35b 76.76 ± 0.75c 3.36a 3.21b 4.09b
APV4 34.13 ± 0.03a 37.61 ± 0.55c 35.85 ± 0.17b 34.50 ± 0.17a 187.01 ± 449.50 ± 357.53 ± 295.58 ±
AHR
3.23a 13.11d 2.56c 3.04b
GSH 3.46 ± 0.23a 6.34 ± 0.60b 5.67 ± 0.18b 6.20 ± 0.24b
Regressions
T-SOD 64.29 ± 1.12a 71.22 ± 0.62b 69.56 ± 0.62b 69.34 ± 0.72b
YPPV = − 0.12X2 + 1.14× + 2
X = 4.94 R = 0.5520 P < 0.05 CAT 8.47 ± 0.08a 13.04 ± 0.12d 11.31 ± 0.30c 10.71 ± 0.22b
38.89
GPX 45.42 ± 1.35a 61.59 ± 1.15c 53.62 ± 1.09b 52.76 ± 0.60b
YAPV = − 0.11X2 + 0.92× + 2
X = 4.06 R = 0.7534 P < 0.05 141.44 ± 212.28 ± 187.36 ± 170.38 ±
35.14 GST
6.52a 7.71d 2.12c 3.47b
YLPV = 1.02× + 67.05 R2 = 0.8911 P < 0.05
GR 9.11 ± 0.56a 14.10 ± 0.33c 12.63 ± 0.59b 11.53 ± 0.36b
LMB = largemouth bass; MAH = methionine hydroxy analogue; PPV = Protein
production value; LPV = Lipid production value; APV = Ash production value. Regressions
1
Values are means ± SE (n = 3 × 6). Values within the same rows having YMDA = 0.04 × 2–0.40× +
X = 4.96 R2 = 0.94 P < 0.05
different superscripts are significantly different (P < 0.05). 1.35
2
PPV (%) = 100 × fish protein gain/total protein intake. YPC = 0.01 × 2–0.13× + 1.41 X = 5.81 R2 = 0.81 P < 0.05
3
LPV (%) = 100 × fish lipid gain/total lipid intake. YASA = − 2.16 × 2 + 22.47× 2
X = 5.20 R = 0.63 P < 0.05
4
APV (%) = 100 × fish ash gain/total ash intake. + 309.74
YAHR = − 9.01 × 2 + 88.90× 2
X = 4.93 R = 0.76 P < 0.05
+ 206.23
YGSH = − 0.07 × 2 + 0.84× + 2
Table 6 3.70
X = 6.44 R = 0.54 P < 0.05
The IW, ISI, IL, and RGL of LMB fed diets with different level of MHA for 84 d1. YT-SOD = − 0.20 × 2 + 2.24×
X = 5.63 R2 = 0.46 P < 0.05
MHA (g/ 0.0 3.0 6.0 9.0 + 64.79
kg) YCAT = − 0.14 × 2 + 1.46× +
X = 5.11 R2 = 0.66 P < 0.05
8.84
IW 1.22 ± 0.07 1.26 ± 0.07 1.24 ± 0.04 1.23 ± 0.11 YGPX = − 0.47 × 2 + 4.73× + 2
ISI2 0.90 ± 0.02 0.91 ± 0.06 0.91 ± 0.08 0.93 ± 0.01 X = 4.99 R = 0.53 P < 0.05
46.98
13.73 ± 14.50 ± 13.83 ± 13.87 ± YGST = − 2.44 × 2 + 24.02×
IL X = 4.92 R2 = 0.59 P < 0.05
0.36a 0.70b 0.79ab 0.84ab + 146.63
3 76.77 ± 80.33 ± 79.62 ± 78.24 ± YGR = − 0.17 × 2 + 1.72× +
RGL X = 5.07 R2 = 0.53 P < 0.05
2.14a 3.63b 2.48ab 4.66ab 9.45
LMB = largemouth bass; MAH = methionine hydroxy analogue; IW = wet in LMB = largemouth bass; MAH = methionine hydroxy analogue; MDA =
testinal weight (g fish− 1); IL = intestinal length (cm fish− 1); ISI = intestosomatic malondialdehyde; PC = protein carbonyl; ASA = anti-superoxide anion; AHR =
index; RGL = relative gut length. anti-hydroxyl radical; GSH = glutathione (mmol g− 1 tissue); T-SOD = total su
1
Values are mean ± SE (n = 3 × 9). Mean values with the different super peroxide dismutase; CAT = catalase; GPX = glutathione peroxidase; GST =
scripts in the same row are significantly different (P < 0.05). glutathione-S-transferase; GR = glutathione reductase.
2
ISI (%) = 100 × wet intestine weight/wet body weight. 1
Values are means ± SE (n = 3 × 6). Values within the same rows having
3
RGL (%) = 100 × intestine length/body length. different superscripts are significantly different (P < 0.05).
b
(Table S1). The effective sequences of all samples were clustered into
b
OTUs with 97% identity. As shown in Fig. 5A and Table 8, the rarefac 3
b
tion curve tended to reach a plateau and the Goods’ coverage of each
c b
b
group reached >98%, which indicated that the sequencing depth was bc
enough in this study. The 105 shared OTUs were detected in all groups, 2 ab
and the number of unique OTUs for the control, 3.0, 6.0, and 9.0 g MHA/ a a
c bc
kg diet groups were 76, 583, 502, and 310, respectively (Fig. 5B). The b b ab a a
a ab
alpha diversity indices of intestinal microbiota were presented in 1
a a a
Table 8. The Chao1 and ACE indices in 3.0 g/kg MHA group showed the
highest values, and fish fed control diet showed the lowest values.
Higher Shannon and Simpson indexes occurred in 3.0 and 6.0 g/kg MHA
0
groups compared to the control group (P < 0.05). Principal coordinates SOD CAT GPx GST GR GCLC
analysis (PCoA) revealed that the bacterial communities of 3.0 and 6.0
g/kg MHA groups clustered together in the third quadrant, while control Fig. 2. Effects of dietary MHA on SOD, CAT, GPX, GST, GR, and GCLC mRNA
expressions in intestine of LMB. Values are means ± SEM (n = 3 × 6), different
and 9.0 g/kg MHA groups clustered in the second and fourth quadrant,
letter denotes significant difference (P < 0.05). MAH = Methionine hydroxy
respectively (Fig. 5C).
analogue; SOD = superoxide dismutase; CAT = catalase; GPX = glutathione
The microbial composition and abundance in intestine of LMB at the
peroxidase; GST = glutathione-S-transferase; GR = glutathione reductase;
phylum level are shown in Fig. 6A and Table S2. The Firmicutes, Bac GCLC = glutamate-cysteine ligase catalytic subunit; LMB = largemouth bass.
teroidetes, Proteobacteria, Cyanobacteria, and Actinobacteria were the
5
Y. Zhao et al. Aquaculture 556 (2022) 738279
Dietary MHA levels (g/kg) abundance of Gemmatimonadetes were significantly increased in 6.0 and
2.0 9.0 g MHA/kg diet groups (P < 0.05).
0.0 3.0 6.0 9.0
At the genus level, the dominant bacterial genera were Unidenti
c fied_Clostridiales, Faecalibacterium, Bifidobacterium, Mangrovibacter,
Relative mRNA expression in intestine
Fig. 4. Effects of dietary MHA on the Keap1 - Nrf2 axis in intestine of LMB. The Nrf2 and Keap1 protein levels were determined by western blot analysis. Equal
loading was monitored with anti-β-actin antibody. Values are means ± SEM (n = 3 × 3), different letter denotes significant difference (P < 0.05). MAH = Methionine
hydroxy analogue; Keap1 = Kelch-like ECH-associated protein 1; Nrf2 = nuclear factor erythroid 2–related factor 2; LMB = largemouth bass.
6
Y. Zhao et al. Aquaculture 556 (2022) 738279
Fig. 5. (A) The rarefaction curve plot of OTUs for the intestinal bacterial samples of LMB fed diets with different level of MHA. M.0.1, M.0.2, M.0.3 are the three
replicates of control group; M.3.1, M.3.2, M.3.3 are the three replicates of 3.0 g/kg group; M.6.1, M.6.2, M.6.3 are the three replicates of 6.0 g/kg group; M.9.1,
M.9.2, M.9.3 are the three replicates of 9.0 g/kg group. (B) Venn diagram demonstrating the distribution of OTUs in LMB fed diets with different level of MHA. M.0,
M.3, M.6, M.9 represents diets containing MHA 0.0, 3.0, 6.0, 9.0 g/kg, respectively. (C) Principal coordinates analysis (PCoA) based on weighted unifrac distances.
M.0, M.3, M.6, M.9 represents diets containing MHA 0.0, 3.0, 6.0, 9.0 g/kg, respectively. OTUs = operational taxonomic units; LMB = largemouth bass; MAH =
Methionine hydroxy analogue.
Fig. 6. Intestinal microbiota composition of LMB fed diets with different level of MHA for 84 d. (A) Relative abundance of intestinal bacteria at the phylum level. (B)
Firmicutes to Bacteroidetes ratio of LMB fed diets with different level of MHA. Values are means ± SEM (n = 3 × 3), different letter denotes significant difference (P <
0.05). (C) Relative abundance of intestinal bacteria at the genus level. Only the top 10 most abundant (based on relative abundance) bacterial phyla and genera were
shown. Other phyla and genera were all assigned as ‘Others’. LMB = largemouth bass; MAH = Methionine hydroxy analogue.
7
Y. Zhao et al. Aquaculture 556 (2022) 738279
being in consistent with the results of Jian carp (Feng et al., 2011). The (Feng et al., 2017). This result shows that dietary MHA increases Nrf2
ASA and AHR activities are two important indicators to assess the total mRNA expression and nuclear protein level, whereas down-regulates
scavenging capacities of O•–2 and OH respectively (Kohen and Nyska,
•
Keap1 mRNA expression and cytosolic protein level in fish intestine.
2016). This study finds that dietary MHA supplementation enhances These results demonstrate that dietary MHA improves intestinal anti
intestinal ASA and AHR activities. Feng et al. also has reported dietary oxidant enzyme-related genes transcription via Keap1/Nrf2 axis. Similar
MHA increased ASA activity in intestine of grass carp (Feng et al., 2011). results have been found in the intestine of grass carp (Pan et al., 2017).
The above results are in agreement with the finding in grass carp in The above results are same to finding in blunt snout bream kidney and
testine by Met (Wu et al., 2017), indicating that MHA plays the similar liver by Met (Ji et al., 2020). Therefore, MHA maybe play the similar
function as Met. Dietary MHA supplementation decreases fish intestinal role as Met in regulating antioxidant enzyme-related genes transcription
lipid oxidation and proteins peroxidation injury via increasing total via Keap1/Nrf2 axis. The Met may regulate the Keap1/Nrf2 axis via the
scavenging capacities of O•–
2 and OH . The scavenging capacities of O2
• •–
PI3K/Akt pathway in blunt snout bream (Ji et al., 2020). However,
and OH• attributes to nonenzymatic compounds content and antioxidant whether MHA regulates the expression of antioxidant enzyme genes via
enzymes activities in fish (Jiang et al., 2016a; Wu et al., 2017), which the PI3K/Akt pathway still needs more research.
are closely associated with the corresponding gene mRNA expressions.
This study finds that dietary MHA supplementation improves the anti 4.3. Dietary MHA regulates intestinal microbiota
oxidant enzymes activities and GSH content. Moreover, T-SOD, CAT,
GPX, GST, and GR activities are positively associated with their corre Intestinal microbiota is one of critical factors to affect nutrient
sponding gene mRNA expressions (Table 9). These results indicate that metabolism and animal growth (Gao et al., 2017; Munaeni et al., 2020;
dietary MHA supplementation could enhance antioxidant enzymes ac Zeng et al., 2017). Less diversity of intestinal microbiota could result in
tivities by up-regulating antioxidant enzymes related gene expressions. the disorder of physiological function, and then cause diseases (Boland
The similar results have been found in blunt snout bream kidney and et al., 2021). The Chao1 and ACE indices and Shannon and Simpson
liver by Met (Ji et al., 2020). These results show that MHA plays the indices are used to characterize microbiota species richness and di
similar function as Met in regulating antioxidant enzymes related gene versity respectively (Peter et al., 2020; Tao et al., 2019; Zheng et al.,
expressions of fish. In addition, the improved GSH content by MHA 2021). The present study shows that dietary MHA supplementation in
might related to GSH de novo synthesis. The MHA could produce creases the microbiota richness and diversity. Study in sow shows that
cysteine, a precursor of GSH, which could then be converted into taurine dietary supplementation with 0.48% Met reduces the bacteria richness
and GSH (Martín-Venegas et al., 2006). The present study also found (Chao1 and ACE) and diversity (Shannon) (Bin et al., 2018). The
that MHA increased GCLC mRNA expressions in LMB. The previous observed differences might contribute to the lower intestinal pH pro
study in piglets showed that Met could be converted to cysteine via duced by MHA. The MHA has a low pH due to its monocarboxylic acid
methylation reactions (Zeitz et al., 2017). But Met decreased GCLC with a hydroxyl group on α carbon. The PCoA is conducted to evaluate
mRNA expressions in rainbow trout O. mykiss (Fontagné-Dicharry et al., differences in microbiota composition (Meng et al., 2021; Tao et al.,
2017). These results indicate that the mechanism of MAH in regulating 2019). In the present study, the PCoA plots show that bacterial com
GCLC gene expression might different from Met in fish. Therefore, the munity form three distinct clusters (control group; 3.0 and 6.0 g MHA/
underlying reason needs further studies. kg diet groups; 9.0 g MHA/kg diet group). This result indicates that MHA
The Nrf2 is a critical transcription factor to regulate antioxidant alters the composition of fish intestinal microbiota. In addition, the
enzyme-related genes transcription (Ji et al., 2020; Suzuki and Yama present study shows that dominant bacterial phyla in intestine are Fir
moto, 2015). The Keap1 represses the translocation of Nrf2 into the micutes, Bacteroidetes, Proteobacteria, Cyanobacteria, and Actinobacteria,
nucleus and Nrf2 mediates gene expression via interacting with Nrf2 which are in accordance with previous studies in LMB (Zhou et al., 2018;
Zhou et al., 2021a), golden pompano Trachinotus ovatus (Tan and Sun,
Table 9 2020), olive flounder Paralichthys olivaceus (Jang et al., 2019), and
Correlation coefficient of some parameters. common carp (Zhang et al., 2021). Nevertheless, these results are
inconsistent with a previous report in Met, which found that Firmicutes,
Independent Dependent Correlation P
parameters parameters coefficients Bacteroidetes and Euryarchaeota were the predominate phyla in sow fed
Met diet (Bin et al., 2018). The differences might be due to the different
PPV 0.862 0.000
FI 0.893 0.000
species or the lower intestinal pH produced by MHA. These dominant
FE 0.995 0.005 bacterial phyla in fish intestine play an important role in nutrient
RGL 0.931 0.069 digestion, absorption, and immune response (Ghanbari et al., 2015; He
MAD − 0.693 0.013 et al., 2021). The Firmicutes/Bacteroidetes ratio is widely used as the
PC − 0.739 0.261
standard of justifying intestinal health (Armougom and Raoult, 2008).
Chao1 0.686 0.014
SGR
ACE 0.684 0.014 This study shows that dietary MHA decreases the Firmicutes/Bacter
Shannon 0.586 0.045 oidetes ratio in intestine of fish. The Proteobacteria is one of the main
Simpson 0.657 0.020 bacteria in the water environment and fish intestines, and it has been
Actinobacteria 0.855 0.145 proved to be strongly correlated with digestion (Kittle et al., 2018; Song
Bifidobacterium 0.988 0.012
Bacillus 0.897 0.103
et al., 2020; Zhou et al., 2018). Some of the beneficial bacteria that
Bacteroides − 0.996 0.004 belong to Proteobacteria can produce polyhydroxy butyrate, which serve
T-SOD SOD mRNA 0.934 0.066 as an energy source for intestine and improve growth of fish (Enomoto
CAT CAT mRNA 0.989 0.011 et al., 2012; Yamazaki et al., 2016). In this study, dietary MHA sup
GPX GPX mRNA 0.882 0.118
plementation increases the abundances of Proteobacteria in intestine of
GR GR mRNA 0.949 0.051
GST GST mRNA 0.959 0.041 fish. Similar results have been reported in sow, where dietary supple
GSH GCLC mRNA 0.649 0.351 mentation with 0.60% Met increases the abundances of Proteobacteria
(Bin et al., 2018). The Actinobacteria, being abundant in fish intestine,
SGR = specific growth rate; T-SOD = total superoxide dismutase; CAT = cata
lase; GPX = glutathione peroxidase; GR = glutathione reductase; GST = gluta produce secondary metabolites, such as alkaloids, lactones, phenazines,
thione-S-transferase; GSH = glutathione; PPV = Protein production value; FI = cyclic peptide compounds, which play a significant role in inhibiting the
food intake; FE = food efficiency; RGL = relative gut length; MDA = malon pathogenic bacteria activity (Wu et al., 2012). In this study, dietary
dialdehyde; PC = protein carbonyl; ACE = abundance-based coverage estimator; MHA increases the abundance of Actinobacteria in the intestine of fish.
GCLC = glutamate-cysteine ligase catalytic subunit. The Cyanobacteria is distributed in a variety of water environment,
8
Y. Zhao et al. Aquaculture 556 (2022) 738279
which is hard to be digested for many fish (Tan and Sun, 2020). Some Funding acquisition. Hui Diao: Methodology, Funding acquisition.
Cyanobacteria can produce a variety of toxins, such as microcystins and Xiao-Lan Wei: Methodology, Funding acquisition. Meng-Jia Zhou:
cyclic lipopeptides, which would lead to damage of digestive organs Methodology, Funding acquisition. Xiao-Qiu Zhou: Conceptualization,
(Amado and Monserrat, 2010; Kang et al., 2012). This study finds that Supervision, Funding acquisition. Jun Jiang: Conceptualization, Su
dietary MHA decreases the abundance of Cyanobacteria in the intestine pervision, Funding acquisition, Writing – review & editing.
of fish. The decreased Cyanobacteria abundance might be attributed to
low pH in intestine. Study on piglet showed that intestine pH was low
ered by dietary MHA supplementation (Kaewtapee et al., 2016). Lower Declaration of Competing Interest
intestinal pH could inhibit Cyanobacteria colonization (Tan and Sun,
2020; Zhou et al., 2018). Recent information on the effect of Met on the The authors declare that no conflict of interest exist.
phylum and genus composition of fish intestinal flora is limited. All
above results suggest that dietary MHA supplementation brings a huge Acknowledgements
alternation to the intestinal microbial community structure in fish,
which potential mechanism requires further studies. This research has received funding from National Key R&D Program
At the genus level, the Bacillus is one of the members of Firmicutes, of China (2019YFD0900200) and National Natural Science Foundation
which has been frequently used as probiotics in aquatic animals (Mujeeb of China (32172987).
Rahiman et al., 2010; Verschuere et al., 2000). The Bacillus secrets many
exoenzymes to aid in the nutritional enhancement of the host (Amoah Appendix A. Supplementary data
et al., 2019; Han et al., 2015). The Bifidobacterium is one of the members
of Actinobacteria, which can improve the integrity of intestinal barrier, Supplementary data to this article can be found online at https://doi.
then enhances the resistance to pathogens (Mokkala et al., 2016). The org/10.1016/j.aquaculture.2022.738279.
Bifidobacterium also has extracellular enzymes that promote digestion of
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11
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he would, he told himself, have been a bear to prevent her dancing
because himself unable, owing to his wounded leg, to dance at all.
Suddenly his spirits rose. He had caught sight of Hopford ... he
was some way off.... Ah, there he was again! Wandering, he
appeared to be in search of some one. And at that instant Hopford
saw him.
“Charlie, for heaven’s sake—I have been hunting for you
everywhere,” Hopford exclaimed when at last they came together.
“An awful thing has happened—it will give you a big shock, but I
implore you not to worry, because I am positive all will come right in
the end. Cora knows about it and is with Yootha now.”
“Yootha? Where is she, Harry? I have been trying to find her for
the last half an hour or more.”
“I can well believe that,” Hopford answered. “Now listen, Charlie,
and don’t get upset. A woman had her pearl necklace stolen to-night,
and the necklace has been found in Yootha’s vanity bag, and so—
well, of course they had to arrest her.”
“Arrest her! Arrest Yootha?”
“Why yes. You see the pearls were found in her possession. Have
you heard about Levi Schomberg, and——”
“Hang Levi Schomberg!” Preston cried out. “What the devil does
Levi Schomberg matter—forgive me, Harry, here, take me to Yootha
at once and let me see the police myself about this ridiculous
trumped-up charge!”
CHAPTER XV.
GATHERING CLOUDS.
Henley week is said to be fine once in eight years, so presumably
it was an eighth year, for the weather was perfect.
Captain Preston, an old blue, had made a point of attending the
historic regatta ever since he had been at school; then had come a
gap, due to the war, and then the regatta had been once more held.
To celebrate the event, also because he thought Yootha would
like it, Preston had this year rented a houseboat which he kept
moored near Maidenhead. Several times before they were engaged
Yootha had spent a day with him on this boat, though he had not
even then made up his mind to propose to her. But now the boat was
moored at Henley, for the regatta week, and he had asked a few of
his friends to come and lunch on board any day they felt inclined to.
It was his intention then to announce his engagement, and to
present them to his future wife.
None of his friends, however, put in an appearance. Some
telegraphed their inability at the last moment to get out of town;
others stayed away without sending an excuse.
Preston was surprised.
“Curious,” he said thoughtfully, as he shook the ashes out of his
pipe about lunch time on the second day of the regatta. “I thought
some of them would turn up to-day. Cooper and Atherton are down
here, I know, because I saw them in a punt together half an hour
ago.”
Yootha, lying back near him in a deck chair, partly concealed by
the overhead awning, did not reply.
“You seem silent to-day, my darling,” he said after a pause. “Is
anything the matter?”
He bent down as he stopped speaking, and peered under the
awning.
The troubled look in her eyes disconcerted him.
“Darling, what is it?” he asked anxiously. “Is something worrying
you?”
“Not worrying me, exactly,” she answered, with rather a wan
smile, “only——”
“Yes? Only what?”
“I think I can guess why your friends have stayed away. It isn’t
hard to guess.”
“Isn’t it? Well, I wish I knew the reason—ah——”
His expression had suddenly changed.
“So now you know,” she went on. “Personally I am not surprised.
You know how people have cold-shouldered me since that dreadful
affair at the ball. Now it has become known we are engaged, people
want less than ever to meet me. Had you been here alone, your
friends would probably all have come to lunch.”
“‘Friends,’ you call them?” Preston exclaimed with a black look.
“They are no longer friends of mine, I can assure you, if they are that
sort.”
“Oh, I don’t blame them,” Yootha answered with a hard smile.
“One has to pay the penalty of notoriety, even though the notoriety
may have come unsought. I dare say I shall live it down,” and she
gave a little shrug.
A tiny boat was sailing past, and the young man seated in the
stern of it hailed Preston.
“Can I come aboard a moment?” he called out. “I want to speak to
you.”
“Come along,” Preston answered, though without enthusiasm, for
the young man was Archie La Planta. Half a minute later the little
boat was alongside.
“I hope I am not intruding,” La Planta said, discovering suddenly,
or pretending to discover, that nobody else besides Yootha was
aboard. “I heard you had a luncheon party. By the way,” he added,
“have I to congratulate you? I heard the news only to-day.”
“Thanks,” Preston said, concealing the annoyance the unlooked-
for intrusion caused him. “Who told you we were engaged?”
“Oh, one or two people. That last race was a fine finish—what?”
“Very. Did you say there was something you wanted to ask me?”
“One or two things. The first is a message from Jessica. She
wants to know if you and Miss Hagerston will come to tea on her
houseboat—it’s that big boat, The Apex, disguised to look like a
warship, with guns and all complete. They’ve a jolly party aboard and
Jessica says she would love to see you both. I was instructed not on
any account to let you say ‘No,’” and he laughed.
Preston did not answer for a moment.
“What do you say, Yootha?” he said at last, with a significant look
which she understood. “Shall we go, or shan’t we?”
“I should like to go,” she replied, taking her cue from his
expression. “Have you had lunch, Mr. La Planta?”
“In point of fact I have not,” he said carelessly, and lit a cigarette.
“Then you had better stay and lunch,” Preston put in. “Others may
drop in presently.”
“Awfully good of you,” La Planta said quietly. “The second thing I
want to ask is whether you happen to have seen Madame Camille
Lenoir of the Metropolitan Secret Agency anywhere about to-day. I
know she is here, with friends, and I want particularly to catch her.”
“I have not noticed her in any of the boats.”
Archie La Planta made a little gesture of annoyance.
“How tiresome,” he said. “Several people have caught sight of her
to-day, but nobody can tell me where she is to be found. By the way,
has the house with the bronze face found anything out as yet about
the pearl necklace?”
“Nothing as yet, but they seem hopeful. They think that in a week
or so they may be able to tell me something.”
“Good. Stothert is not an optimist, and would not have told you
that unless he had good reason to.”
He turned to Yootha.
“By the way, I have not seen you since the ball,” he said. “And for
that matter I didn’t see you at the ball, because I couldn’t identify
you. That was a rotten experience you had—perfectly disgraceful to
treat you as they did. I hear it made you quite ill, and I am not
surprised. I hope that by now you have quite recovered?”
“Thank you,” she answered, and in spite of her effort to speak
naturally she could not prevent a certain coldness from betraying
itself in her voice.
“Yes, I have recovered—though I don’t think all my friends have!”
“Indeed? I think I don’t follow you.”
“Oh, it’s of no consequence,” she replied, flushing slightly; then
she changed the subject.
Jessica was surprisingly genial and friendly when she greeted
Yootha and Captain Preston on her houseboat a couple of hours
later. Preston had availed himself of her invitation for a reason which
he had not yet confided to Yootha, though, had he known it, the
same reason had prompted Yootha to ask La Planta if he had
lunched.
They knew that in spite of the olive branch now held out by
Jessica, at heart Jessica’s dislike of them both had become, if
anything, intensified since the affair at the ball, and that her hatred of
Cora too had increased. Though not addicted to crediting gossip, so
many little remarks of Jessica’s concerning themselves had been
repeated to them by different people that they could not turn an
entirely deaf ear.
All sorts of well-known people were on Jessica’s houseboat.
Some Yootha had met, and to many of the remainder she and
Preston were introduced. Indeed so friendly did everybody appear to
be that presently she began to feel quite happy and at home—the
reverse of what she and Preston had anticipated.
And of all aboard, none made himself more agreeable to Yootha
and to Captain Preston than Archie La Planta. If anything, he rather
overdid it, for he took the trouble to present several people whom
they found extremely boring. They had been aboard perhaps half an
hour, when Yootha suddenly heard her name spoken, and, turning,
found herself face to face with a dark, very intelligent-looking man
approaching middle age.
“Let me introduce Doctor Johnson,” Stapleton said. “Johnson—
Miss Hagerston.”
The doctor looked at her keenly, smiling as he raised his hat.
“Our common friend, Captain Preston, has several times
mentioned your name,” he said. “Is he with you to-day?”
“Why, yes,” Yootha answered. “He was here a minute ago. Have
you only just come aboard?”
“No, I have been here the whole afternoon, but for the last hour I
have been what I suppose is called ‘’tween decks,’ settling the
nation’s affairs with some of my cronies whom I don’t often have an
opportunity of meeting,” and he smiled. “Stapleton tells me, Miss
Hagerston, that you and Preston are engaged. May I be allowed to
offer my congratulations? I should like to congratulate you both very
sincerely.”
Yootha colored as she looked over his shoulder.
“Thank you so much,” she said. “I feel as if we had met before;
Charlie has told me so much about you.”
“Well, though I have known him quite a short time, you will forgive
my saying that I like him immensely. Yet, but for the unfortunate affair
of that man’s death the other night, I suppose I should not have had
the pleasure of meeting him, or possibly you. Ah, here he comes.”
For five minutes or more the three remained in conversation,
though their talk was mostly commonplace. There were subjects all
three would have liked to broach, but to have done so amid that
entourage would have been impolitic.
“Yootha and I are dining tête-à-tête on my houseboat,” Preston
said after a while. “If you are not engaged, couldn’t you join us—if
you will take potluck? Do say you will, Johnson.”
Johnson reflected for some moments.
“I should like to very much,” he said at last. “It is most kind of
you.”
“Capital! Then that is arranged.”
A diversion was created by the approach of a motor-launch with a
party of masked entertainers, while the string quartette in the bows
played a popular air. As it came near it slowed down, then stopped
alongside.
“Oh, those people are splendid,” Jessica exclaimed. “They were
here yesterday and played for us during lunch. Louie,” she turned to
Stapleton, “make them give us an entertainment now.”
The entertainment lasted a long time, so that Preston and Yootha
were unable to leave the houseboat, as they had been about to do
when the launch came in sight. When at last the entertainment
ended they sought out their hostess.
“But surely you are not going?” Jessica exclaimed, holding
Yootha’s hand. “We are only just beginning to enjoy ourselves! Can’t
you both stay to dinner? We want to drink your health, you know,”
and she laughed in her deep and musical voice.
“So good of you,” Yootha answered, though all the while her
instinct told her that beneath this show of friendship and hospitality
there lurked some sinister motive, “but we have a friend dining with
us on our boat.”
“Ah, in that case I suppose there is nothing further to be said,”
Jessica replied. “But I am very disappointed. Come to lunch to-
morrow, will you? And you too, Captain Preston, make her bring you
with her.”
But Preston excused himself on the plea that he expected friends
to lunch.
“Sometimes, though, friends don’t turn up,” Jessica said
inconsequently. “If they don’t, mind you come, both of you.”
A few minutes after they had gone a man rowed up rapidly in a
dinghy.
“Can I speak to Captain Preston, please?” he asked. “I am his
servant. I have just come from his house-boat.”
He had addressed La Planta, who, leaning against the rail, had
been watching him approach.
“Is it anything important?” La Planta inquired, eyeing the man
coldly.
“It is.”
“Well, Captain Preston and Miss Hagerston have just left. You
must have passed them.”
“Have they gone back to the boat?”
“I expect so. They went down stream.”
Borne along the water came the strains of a revue waltz played
further down the river by the string quartette.
Preston’s servant pulled the dinghy round, then started to row
back.
And La Planta, blowing rings of cigarette smoke, watched him,
with a look of amusement, growing smaller and smaller in the
distance.
At last he straightened himself. Most of the guests had now left.
Barely a dozen remained. Some one touched his elbow, and he
turned.
“Well?” Stapleton said.
La Planta nodded.
“Quite satisfactory,” he answered.
“And when will he receive it?”
La Planta glanced down at his wrist-watch.
“He may have got it already. I am glad that fellow missed him,
though I can’t think how he managed to. What fools people like
Preston and that girl of his and Cora Hartsilver and the rest of them
are to pit their wits against ours!”
“Preston is no fool, Archie. Nor, for that matter, is Cora.”
But La Planta made no reply. Instead, he shrugged his shoulders,
then tossed his cigarette into the water with a gesture of contempt.
CHAPTER XVII.