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Impacts of Dietary Protein and Niacin Deficiency on
Reproduction Performance, Body Growth, and Gut Microbiota
of Female Hamsters (Tscherskia triton) and Their Offspring
Jidong Zhao,a,b Wei Lu,aShuli Huang,aYvon Le Maho,c,d Caroline Habold,cZhibin Zhanga,e
a
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People’s Republic of China
b
Shaanxi Key Laboratory of Qinling Ecological Security, Shaanxi Institute of Zoology, Xi'an, People’s Republic of China
c
University of Strasbourg, CNRS, IPHC, UMR 7178, Strasbourg, France
d
Scientific Centre of Monaco, Monaco Principality, Monaco
e
CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, People’s Republic of China
ABSTRACT Food resources are vital for animals to survive, and gut microbiota play
an essential role in transferring nutritional materials into functional metabolites for
hosts. Although the fact that diet affects host microbiota is well known, its impacts
on offspring remain unclear. In this study, we assessed the effects of low-protein
and niacin-deficient diets on reproduction performance, body growth, and gut
microbiota of greater long-tailed hamsters (Tscherskia triton) under laboratory condi-
tions. We found that maternal low-protein diet (not niacin deficiency) had a signifi-
cant negative effect on reproduction performance of female hamsters (longer mating
latency with males and smaller litter size) and body growth (lower body weight) of
both female hamsters and their offspring. Both protein- and niacin-deficient diets
showed significant maternal effects on the microbial community in the offspring. A
maternal low-protein diet (not niacin deficiency) significantly reduced the abundance
of major bacterial taxa producing short-chain fatty acids, increased the abundance of
probiotic taxa, and altered microbial function in the offspring. The negative effects
of maternal nutritional deficiency on gut microbiota are more pronounced in the
protein group than the niacin group and in offspring more than in female ham-
sters. Our results suggest that a low-protein diet could alter gut microbiota in
animals, which may result in negative impacts on their fitness. It is necessary to
conduct further analysis to reveal the roles of nutrition, as well as its interaction
with gut microbes, in affecting fitness of greater long-tailed hamsters under field
conditions.
IMPORTANCE Gut microbes are known to be essential for hosts to digest food and
absorb nutrients. Currently, it is still unclear how maternal nutrient deficiency affects the
fitness of animals by its effect on gut microbes. Here, we evaluated the effects of protein-
and niacin-deficient diets on mating behavior, reproduction, body growth, and gut micro-
biota of both mothers and offspring of the greater long-tailed hamster (Tscherskia triton)
under laboratory conditions. We found that a low-protein diet significantly reduced
maternal reproduction performance and body growth of both mothers and their off-
spring. Both protein and niacin deficiencies showed significant maternal effects on the
microbial community of the offspring. Our results hint that nutritional deficiency may be
a potential factor in causing the observed sustained population decline of the greater
long-tailed hamsters due to intensified monoculture in the North China Plain, and this
needs further field investigation.
KEYWORDS cropland monoculture, greater long-tailed hamster, gut microbiota, low-
protein diet, niacin deficiency
Editor Kevin R. Theis, Wayne State University
Ad Hoc Peer Reviewer Dominik Schmid
Copyright © 2022 Zhao et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Zhibin Zhang,
zhangzb@ioz.ac.cn.
The authors declare no conflict of interest.
Received 16 January 2022
Accepted 10 October 2022
Published 1 November 2022
November/December 2022 Volume 10 Issue 6 10.1128/spectrum.00157-22 1
RESEARCH ARTICLE
Anutritionally balanced diet is vital for maintaining biological processes and the sur-
vival of all organisms. Nutritional deficiency can reduce the fitness of animals by
disrupting behavior and prohibiting growth or reproduction. Amino acids are essential
for protein biosynthesis and regulation of cell signaling and metabolic pathways. Low
protein intake has been shown to be associated with increased inflammation risk (1),
prohibition of postnatal body growth (2), and reduction of fertility of adult females (3)
and offspring (4) in many murine species. Niacin (i.e., vitamin B
3
, or nicotinamide) is
one of the water-soluble vitamins that are involved in various biological functions for
maintenance of normal growth and reproduction of animals. Deficiency in dietary nia-
cin may disrupt the maternal behavior of hamsters (3) and increase risk of disease in
humans, such as pellagra and diarrhea (5) or colitis (6). Although either a low-protein
diet (LPD) (2, 7–9) or a niacin-deficient (niacin
2
) diet (10–12) can cause abnormality in
reproduction of female animals, the interaction of dietary protein and niacin deficiency
has not been fully investigated.
Nutrient deficiency may be one of potential reasons for population decline in some
wild animal species. Intensified monoculture is suggested to be one of the key factors
that threatens rodent species in cropland (13–15). Crop monoculture practices may
influence the diet structure of cropland wildlife by reducing overall biodiversity in the
habitat (3, 15, 16). For instance, the European hamster was once widely distributed in
Eurasian states, but has now become endangered worldwide. Deficiencies in dietary
protein and niacin, resulting from wheat and maize monoculture, may have contrib-
uted to the reproductive failure of European hamsters, thus accelerating the decline of
their wild population (3). However, the maternal effects of protein and niacin defi-
ciency on offspring due to gut microbial imbalances remain unclear.
Gut microbiota are essential in maintaining normal function and health of hosts: e.g.,
food digestion, immune response, metabolic homeostasis, and other bodily processes (17–
20). Access to food resources is a key factor in shaping homeostasis of the gut microbial
community in animals (21, 22), while disturbances in gut microbes may detrimentally affect
gut metabolic function (18, 23–25). Nutrient deficiency may disrupt gut microbiota, also
reducing fitness of hosts and their offspring. Dietary protein deficiency can alter the micro-
bial community (26–29), but available results are often contradictory (28). Niacin deficiency
can also affect gut microbes, as well as the health of both hosts and their offspring animals
(10–12, 30). Maternal gut microbiota may affect offspring during vaginal delivery and
breastfeeding (31, 32), but the effects of maternal nutrient deficiency on offspring need fur-
ther investigation.
The greater long-tailed hamster (Tscherskia triton) is widely distributed in north
China and some parts of Russia and North and South Korea. It was previously a pre-
dominant pest rodent species in the farmlands of the North and Northeast China Plain
(33). Recently, sustained population decline of this species has been reported (34–36).
A sympatric species, the Chinese striped hamster (Cricetulus barabensis), is also suffer-
ing from a similar sustained population decline (37). From 1981 to 2015, areas in the
North China Plain used for planting soybeans and cotton decreased, while areas for
maize increased (38). Similar to the case of European hamsters (3), dietary changes
resulting from planting a monoculture may have contributed to the observed popula-
tion decline of T. triton, but this hypothesis has not been tested.
The purpose of this study was to examine the effects of maternal protein and niacin defi-
ciency on body growth, reproduction performance, and gut microbiota of the greater long-
tailed hamsters under laboratory conditions. We designed a 2- by 2-factor experiment, making
up four diet treatment groups with combinations of low-protein diet (LPD) or normal-protein
diet (NPD) and niacin-supplemented (niacin
1
)orniacin-deficient (niacin
2
)dietforfemale
hamsters: (i) the NPD-niacin
1
group, (ii) LPD-niacin
1
group, (iii) NPD-niacin
2
group, and (iv)
LPD-niacin
2
group (for details, see Materials and Methods and Fig. 1). Adult female hamsters
werefedwithstandardandmodified AIN93G rodent chow containing the above four differ-
ent diets for 1 month; body weight was measured, and feces were collected in order to ana-
lyze gut microbes. Then, the females were assigned to cohabit with normal adult males for
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
November/December 2022 Volume 10 Issue 6 10.1128/spectrum.00157-22 2
14 days. During the cohabitation tests (each test lasted 1 day), we measured the mating
behaviors of the female hamsters, including mount latency of the female (time needed for a
female to have the first successful mating with a male), mount frequency (number of copula-
tions within 15 min after cohabitation with an adult male), and number of cohabitation tests
(number of cohabitation tests needed for a maternal female to successfully mate with an adult
male). After successful mating, females were housed individually until giving birth, and their lit-
ter sizes were measured, body weights of pups at weaning were measured, and feces were
collected to analyze gut microbes. The effects of maternal protein and niacin deficiency on
body growth, reproduction performance, and gut microbiota of the greater long-tailed ham-
sters were statistically tested. We focused on testing for the following two hypotheses: (i)
maternal low protein and niacin deficiency would have a negative effect on body growth and
reproductionperformanceoffemalehamstersandthebodygrowthofoffspring,and(ii)
maternal low protein and niacin deficiency would alter gut microbes of both maternal and off-
spring hamsters. If these hypotheses held, it would provide us with important implications
that nutritional deficiency may play a potential role in population decline of T. triton under the
intensified monoculture in the North China Plain, and more work needs to be done to confirm
this potential issue.
RESULTS
Effects of maternal protein- and niacin-deficient diets on body weight of
female hamsters and their offspring. We found there was no overall significant effect
of protein (F=2.56;P= 0.116)- and niacin (F=0.82;P=0.371)-deficient diets on body
weight of female hamsters during the study period (see Table S2 in the supplemental
material). We found there was a significant interaction effect between protein diet and
sampling time (F= 25.41; P,0.0001) (Fig. 2A); the body weight of female hamsters in
the maternal low-protein diet (LPD) groups was significantly lower than that in the
maternal normal-protein diet (NPD) groups at days 14, 18, 22, and 30 (Fig. 2A). Maternal
LPD significantly decreased offspring body weight during the weaning period (P,
0.0001) (Fig. 2B and Table S2). We did not find a significant effect of niacin deficiency on
body weight for both female hamsters and offspring (P.0.05) (Table S2).
Effects of protein- and niacin-deficient diets on reproduction of female ham-
sters. We found maternal LPD significantly increased the number of cohabitation tests
(one test per day) needed for a female to successfully mate with an adult male com-
pared to NPD (P,0.001) (Fig. 2C and Table S3). There was no such significant effect of
niacin deficiency (P,0.05) (Table S3). We found maternal LPD significantly increased
the mount latency of females compared to that in the NPD group (P,0.001) (Fig. 2D
FIG 1 Schematic overview of the experimental design. (A and C) Experimental time course for (A) adult females
(mother hamsters) and (C) adult males (for mates of female hamsters) of greater long-tailed hamsters. Adult female
hamsters (n= 51) were fed with standard and modified AIN93G rodent chow of four different diets in Table 2, and
adult males (n= 51) were fed with only standard AIN93G rodent chow. (B) Cohabitation tests for breeding and
measuring the mating behaviors of adult female hamsters. For details, see Materials and Methods.
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
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and Table S3). However, there was no significant difference in mount frequencies of
female hamsters with NPD and LPD (P.0.05; NPD, 6.41 60.49; LPD, 7.11 60.54) or
niacin
1
and niacin
2
(P.0.05; niacin
1
, 7.07 60.67; niacin
2
, 6.55 60.41) (Table S3). An
LPD significantly decreased litter size in female hamsters (P,0.001) (Fig. 2E and Table
S3). There was no significant difference in sex ratio (male/female) in offspring with NPD
and LPD (P.0.05; NPD, 2.19; LPD, 1.63) or niacin
1
and niacin
2
(P.0.05; niacin
1
, 2.64;
niacin
2
, 1.46) (Table S3). We observed one case of infanticide in a maternal female in
the niacin
2
diet group (Table S1).
Effects of protein- and niacin-deficient diets on gut microbiota of female ham-
sters and their offspring. We found a significant effect of protein-deficient diet on the
gut microbial community of female hamsters during the 1-month adaptation (P=
0.002) (Table 1 and Fig. 3A), but there was no such significant effect of a niacin-defi-
cient diet on female hamsters (P= 0.363) (Table 2 and Fig. 3A). A maternal protein-defi-
cient diet significantly altered the gut microbial community of offspring at weaning
(P= 0.001) (Table 1 and Fig. 3B), while the niacin-deficient diet failed to affect the over-
all gut microbial community of offspring hamsters (P= 0.207) (Table 1 and Fig. 3B).
b
dispersion analysis indicated maternal LPD significantly reduced interindividual vari-
ation in the gut microbial community of offspring hamsters compared to the maternal
NDP diet (P= 0.001) (Fig. 3B and Table 1).
We found no significant effect (P.0.05) of protein-deficient or niacin-deficient diets on
a
diversity indices of female hamsters (Table S4). However, we found that maternal LPD sig-
nificantly decreased the Shannon diversity, Chao1 index, and observed features in offspring
hamsters (Fig. 3C and Table S4), while the maternal niacin-deficient diet significantly
increased the Shannon diversity, Chao1 index, and observed features in offspring hamsters
(Fig. 3D and Table S4). Both maternal LPD (P= 0.500) and maternal niacin
2
(P= 0.543) had
no significant effect on the phylogenetic diversity index of gut microbiota in hamster
FIG 2 Significant effects of protein diet on body weight, mating behaviors, and reproduction of
female hamsters and body weight of offspring hamsters. (A) Body weight change (mean 6standard
error of the mean [SEM]) of female hamsters under normal-protein diet (NPD) and low-protein diet
(LPD); (B) body weight of offspring hamsters at weaning period; (C) number of cohabitation tests for
successful mating of a female hamster with a male hamster; (D) mount latency of female hamsters at
their first copulation; (E) litter size of female hamsters. *,P,0.05; **,P,0.01; ***,P,0.001.
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
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offspring (Table S4). Sex and litter size showed nonsignificant effects on
a
diversity in the off-
spring (all Pvalues of .0.05). Fecal microbiota of both female hamsters and their offspring
consisted of nine predominant phyla, including Firmicutes,Bacteroidetes,TM7,Proteobacteria,
Verrucomicrobia,Actinobacteria,Tenericutes,Spirochaetes,andCyanobacteria (Fig. 4).
Analysis with ANCOMBC (analysis of compositions of microbiomes with bias correction)
indicated that there was no significant differential taxa at the phylum level in female ham-
sters between protein- and niacin-deficient diets (adjusted P.0.05). However, we found
theabsoluteabundanceofProteobacteria (adjusted P,0.0001) and Spirochaetes (adjusted
P,0.0001) was significantly decreased, while that of Actinobaceria (adjusted P,0.0001)
in the offspring was significantly increased in the maternal LPD group. There was no signifi-
cant such difference of phyla (adjusted P.0.05) between the maternal niacin
1
and
niacin
2
diet groups. A protein diet significantly altered the absolute abundance of 27 gen-
era from Firmicutes,Bacteroidetes,Proteobacteria,Spirochaetes,Actinobacteria,Tenericutes,
Deferribacteres,andFusobacteria of female hamsters (Fig. 5A and Table S5) and 41 genera
from Firmicutes,Bacteroidetes,Proteobacteria,Spirochaetes,Actinobacteria,Tenericutes,
Cyanobacteria,andDeferribacteres in offspring (Fig. 5C and Table S6). Maternal LPD signifi-
cantly decreased the absolute abundance of the genera Dorea,Desulfovibrio,Helicobacter,
Sporobacter,Treponema,andRuminococcus but increased the absolute abundance of
Lactobacillus and Bifidobacteria in gut microbiota of the offspring (Fig. 5C and Table S6).
Absolute abundance of the genera Anaeroplasma,Gemmiger,andSpirochaeta showed the
same response in both female and offspring hamsters to the LPD (Fig. 5A and C and Tables
S5 and S6). Also, niacin
2
significantly decreased the absolute abundance of the genera
Macrococcus,Mucispirillum,Clostridium,Arthrobacter,Alkanindiges,andHespellia,butit
increased the abundance of the genera Spirochaeta and Proteiniborus (Fig. 5B and Table S7).
Analysis using the Tax4Fun2 algorithm indicated that neither a protein- nor niacin-defi-
cientdietsignificantly altered the microbial functional orthologs (NPD versus LPD, P.
0.05; niacin
1
versus niacin
2
,P.0.05) (Table S8) and KEGG pathways (NPD versus LPD, P
.0.05; niacin
1
versus niacin
2
,P.0.05) (Table S8) in female hamsters. However, microbial
functional orthologs in offspring were significantly changed by maternal LPD (P= 0.021)
(Fig. 6 and Table S8), but the KEGG pathway was not significantly affected (P= 0.073). We
found no significant difference in functional orthologs (P.0.05) or KEGG pathways (P.
0.05) between niacin diet groups (Table S8). LEfSe (linear discriminant analysis [LDA] effect
size) analysis revealed 53 enriched functional orthologs and 32 enriched pathways (KEGG
level 1) in offspring hamsters (Fig. 6 and Tables S9 and S10), and most of the altered func-
tions and pathways are associated with protein metabolism.
DISCUSSION
Itisknownthatproteinorniacindeficiencies may negatively affect the body growth
and reproduction of animals, but their maternal impacts on fitness and gut microbiota of
TABLE 1 Effects of protein and niacin diets on interindividual variation and composition of
gut microbiota of female and offspring hamsters
a
Parameter
Female hamster Offspring hamster
For pseudo-F
b
Pvalue R
2
For pseudo-F
b
Pvalue R
2
b
dispersion
Protein (NPD vs LPD) 0.43 0.503 25.03 0.001
Niacin (niacin
1
vs niacin
2
) 0.0012 0.974 0.90 0.359
PERMANOVA
Protein (NPD vs LPD) 1.87 0.002 0.052 2.68 0.001 0.081
Niacin (niacin
1
vs niacin
2
) 1.04 0.363 0.029 1.11 0.207 0.039
Protein niacin 1.09 0.262 0.030 0.97 0.584 0.027
Dam’s ID 3.80 0.001 0.439
a
NPD, normal-protein diet; LPD, low-protein diet; niacin
1
, niacin-supplemented diet; niacin
2
, niacin-deficient
diet.
b
Fvalues are shown for
b
dispersion parameters, and pseudo-Fvalues are shown for PERMANOVA parameters.
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
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offspring are not fully investigated. In this study, we found maternal LPD, but not niacin
2
diet, significantly and negatively affected mating behavior, reproduction, and body growth
of maternal greater long-tailed hamsters (T. triton) and body growth of their offspring.
However, both protein and niacin deficiencies showed significant maternal effects on the
microbial community of their offspring. Maternal LPD (not niacin
2
diet) significantly altered
the microbial functions of offspring. Our results generally support the two hypotheses,
with additional findings that protein deficiency has a more pronounced effect than niacin
deficiency and the malnutrition effect is greater in offspring than in female hamsters.
Effects of nutritional deficiency on hamster body growth. Administration of a
nutritional deficiency diet during pregnancy and lactation can decrease body weight of
both maternal females and their offspring. A maternal LPD may restrict postnatal body
growth (2), promote inflammation risk (1), and impair the fertility of offspring in later life (4)
in laboratory rodent species. Niacin supplements may attenuate the weight loss in rats
FIG 3 Differences in gut microbial diversity of hamsters under different protein diets and niacin diets. (A and
B) Aitchison distance PCoA for female hamsters (A) and offspring hamsters (B). (C and D)
a
diversity indices of
offspring for protein diet groups (C) and niacin diet groups (D). *,P,0.05; **,P,0.01; ***,P,0.001.
Abbreviations: NPD, normal-protein diet; LPD, low-protein diet; Niacin
1
, niacin-supplemented diet; Niacin
2
,
niacin-deficient diet.
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
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with severe colitis (6). In this study, we found a maternal LPD significantly reduced
body weight of offspring at weaning, with an almost 50% decrease in body weight,
compared to the niacin-deficientdiet(Fig.2B),whichisconsistentwithprevious
findings (2, 7, 8). The effects of niacin deficiency on body weight were not observed
in our study; however, we found one case of maternal infanticide in the niacin-defi-
cient group, which is similar to the finding in European hamsters fed a maize-based
diet (3). It is not clear whether the observed infanticide behavior was associated with
niacin deficiency or not in our study due to the observation of only a single case.
Decreased body weight at weaning is likely caused by a decrease in milk production or
composition change (9). However, the roles of milk production or its composition change
in offspring are not clear because we did not have these data. Because the body weight
of female hamsters was also reduced in LPD groups, it is likely that protein levels were
also decreased in maternal milk. In future studies, it is necessary to examine the milk pro-
duction and nutritional changes in maternal animals and identify how these changes
affect their offspring.
Effects of nutritional deficiency on reproduction performance of hamsters.
Maternal nutritional deficiency may negatively affect reproduction performance of ani-
mals. An LPD may induce cognition decline and anxiety-like behavior in mice (39).
Litter size was significantly affected by an LPD in our study, which is a result similar to
FIG 4 Difference of the relative abundances of the dominant gut microbial phyla in female and offspring hamsters between different
protein diet (A) and niacin diet (B) groups at the phylum level. Abbreviations: NPD, normal-protein diet; LPD, low-protein diet;
Niacin
1
, niacin-supplemented diet; Niacin
2
, niacin-deficient diet.
TABLE 2 Nutritional parameters in four protein and niacin diet groups for female hamsters in
a 2- by 2-factor experiment
a
Nutritional parameter
Result for diet group:
NPD-niacin
+
NPD-niacin
2
LPD-niacin
+
LPD-niacin
2
Protein, % 22.2 22.2 8.8 8.8
Fat, % 6.8 6.8 6.9 6.9
Niacin, mg/kg 30.0 0 30.0 0
Carbohydrate, % 55.5 55.5 67.5 67.5
Total energy (kcal/kg) 3.72 3.72 3.68 3.68
a
The diet formula was modified based on standard AIN93G rodent chow. Standard (basal) AIN93G rodent chow
contained normal protein with niacin supplement. Abbreviations: NPD, normal-protein diet; LPD, low-protein
diet; niacin
1
, niacin-supplemented diet; niacin
2
, niacin-deficient diet. Differences in nutrients are highlighted in
boldface.
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those from studies of the European hamster (3). In this study, we found that a maternal
LPD significantly and negatively affected mating behavior, reproduction, and body
growth of female hamsters. A maternal LPD significantly increased the number of
cohabitation tests for a female to successfully mate with a male and mount latency but
decreased litter size (Fig. 2C, D, and E). We speculate that a maternal LPD may impair
the development of the nervous system and then disrupt the cognitive ability of
female hamsters, which is essential for mating. In our study, we did not find the signifi-
cant effect of niacin deficiency on reproduction performance of female hamsters,
which is different from findings in the European hamster (3). The difference may be
FIG 5 Significant differential abundance of fecal microbial genera (log fold change). (A) Normal-protein diet (NPD) versus low-protein diet (LPD) in fecal
microbiota of female hamster; (B) niacin-supplemented diet (Niacin
1
) versus niacin-deficient diet (Niacin
2
) in fecal microbiota of offspring hamster; (C) NPD
versus LPD in fecal microbiota of offspring hamster.
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November/December 2022 Volume 10 Issue 6 10.1128/spectrum.00157-22 8
caused by species due to a difference in physiological demands for niacin or in the bio-
synthesis of niacin during the perinatal period (40).
Effects of protein deficiency on the gut microbial community of hamsters. An
LPD could disrupt homeostasis of gut microbiota and result in a set of disorders (26, 28). A
few previous studies have evaluated the response of the gut microbiome to dietary protein
shift (29). However, results representing the effects of protein on the gut microbiome are
not consistent, probably due to differences in protein concentration or protein source or
species (28, 41–43). An LPD may increase
a
diversity of microbiota in rodents (41) but
decrease
a
diversity in insects (42), while in a cohort study in humans, no significant effect
of dietary protein on microbial
a
diversity was found (43). Nonetheless, lower diversity of
gut microbes in humans is found to be linked to many diseases, such as obesity and
inflammatory bowel disease (IBD) (19). In this study, we found maternal protein deficiency
significantly affected the microbial community in both female hamsters and their offspring
(Table 1 and Fig. 3), and
a
diversity of offspring was significantly reduced (Fig. 3C), which
was associated with their metabolic function pathways (described below). Because gut
microbes play an essential role in normal function and health of hosts (17–19), the LPD-
altered microbes appear to have a negative effect on body growth in offspring, but this
needs further investigation (44).
Based on results of predicted pathways (Fig. 6C and see Table S9 in the supplemental
material), we observed a significant decrease in protein metabolism (phenylalanine, tyro-
sine, tryptophan, valine, leucine and isoleucine biosynthesis, and histidine metabolism),
FIG 6 Differences in functional orthologs and KEGG pathways of the fecal microbial community in offspring hamsters between maternal normal-protein
diet (NPD) and low-protein diet (LPD) groups by using PCoA. (A) Predicted KO (KEGG Orthology) functional orthologs; (B) KEGG pathways. (C); differential
KEGG pathways between NPD and LPD. Negative and positive LDA scores represent those pathways enriched in offspring hamsters of maternal LPD and
NPD groups, respectively. Abbreviations: Niacin
1
, niacin-supplemented diet; Niacin
2
, niacin-deficient diet.
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protein machinery (ABC transporters), signal transduction (two-component system), and
cell motility (bacterial chemotaxis and flagellar assembly) in maternal LPD offspring. These
pathway depletions may be associated with the reduction of bacterial diversity in the off-
spring. However, no such difference was found in female hamsters, suggesting the effects
of protein deficiency were more pronounced in the early development of offspring during
pregnancy or the lactation period. The effects of LPD on microbial structure in female ham-
sters could be partially weakened by other factors, such as a compensatory decrease in
muscle breakdown, increased food intake, or strengthened proteolytic activity in the large
intestine (28, 45). Increasing evidence suggests that mother gut microbiota is important in
fetus development and breastfeeding (31, 32, 46). The deterioration of milk quality in
female hamsters may also directly influence gut microbiota in offspring, which may result
in a decline of carbohydrate and amino acid metabolism processes as observed in this
study (Fig. 6C and Table S9). Further studies based on manipulation of gut microbiota are
needed to elucidate whether the alteration seen in the offspring’smicrobiotawascaused
by the mother hamsters’microbiota.
Lactobacillus and Bifidobacterium account for ;17.20% and 0.60% of all microbes
found in the total fecal samples in our study. Many species in these genera were recog-
nized as probiotics to prevent or treat gastrointestinal diseases or alleviate behavioral
disorders (47, 48). Breastfeeding is the key postnatal link between mothers and neo-
nates and drives the microbial colonization (49, 50). A previous study reported that an
LPD in mice after weaning could reduce the mucosal colonization of Lactobacillus and
inhibit its recognition by IgA (51). We found maternal LPD significantly increased abun-
dance in Lactobacillus and Bifidobacterium in the offspring (Fig. 5C and Table S6). The
difference may be caused by difference in milk intake of the offspring during the lacta-
tion period, which needs further investigation.
Wefoundabundanceofthetopsixtaxa,includingTreponema,Desulfovibrio,Sporobacter,
Helicobacter,Dorea,andRuminococcus, was decreased but probiotics were enriched in off-
spring of maternal LPD group (Fig. 5C and Table S6). No such effects were observed in off-
spring of maternal niacin
2
diet group. These observations suggested that gut microbiota of
hamsters was more sensitive to dietary protein deficiency than to niacin deficiency. Lower
abundance of Desulfovibrio was correlated with lower body mass index (BMI) (52) and birth
weight (53) in humans, which is consistent with our observation that lower body weight of
offspring was also associated with lower abundance of Desulfovibrio. Moreover, we observed
that genera with a larger change in abundance like Unclassified Lachnospiraceae,Dorea,and
Ruminococcus were fermented bacteria (Fig. 5C and Table S6), which contain various short-
chain fatty acid (SCFA)-producing species (54–58). SCFAs are primary end products fermented
from dietary fibers by gut microbiota, which play an important role in modulating gut homeo-
stasis, immune function, and even gut-brain communication (56, 58). Disturbance of SCFA pro-
ducers could be caused by the decreased nutritional content in offspring, which may detri-
mentally impact the colon health of animals (47, 57). Therefore, reduction of these important
SCFA-producing microbes in the LPD group may impose negative effects on both female
hamsters and their offspring.
Effects of niacin deficiency on gut microbiota of hamsters. Niacin (also called vita-
min B
3
), can be found in several forms, like nicotinamide and nicotinic acid, and is required
in multilevel cellular processes. Mammals are able to absorb niacin from food or biosynthe-
size it from tryptophan (5, 40). Niacin can exert beneficial effects on maintaining gut health
(11, 30). However, the effects of niacin on an offspring’s gut microbiota have rarely been
investigated. In this study, we found that maternal niacin-deficient diets significantly
altered the gut microbial community in offspring hamsters (but not in adult female ham-
sters). It is likely that niacin is synthesized from other substrates, such as tryptophan (maize
starch and casein contains low levels of tryptophan), in the diet of maternal animals; there-
fore, the reproduction and gut microbiota are less affected by niacin deficiency (40).
Besides, maternal niacin deficiency may impact the gut microbial community through
reduction in milk yield as demonstrated in another study (10). We observed that niacin
deficiency significantly increased
a
diversity indices (Chao1 and observed features) of
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November/December 2022 Volume 10 Issue 6 10.1128/spectrum.00157-22 10
offspring hamsters (Fig. 3D), which is contradictory to a previous study (59). We speculate
that, in our study, maternal niacin deficiency may have stimulated the biosynthesis of nia-
cin, which is involved in multiple bacterium species.
Limitations of this study. This study suffers several limitations that restrict the inter-
pretation of some results. First, although nutrition deficiency may reduce the fitness of ham-
sters under laboratory conditions, the causal links between nutrition deficiency and sus-
tained population decline of greater long-tailed hamsters under field conditions are still
unclear. Thus, we are not sure if the observed negative effects of malnutrition on hamsters
in the laboratory are applicable to field conditions. Future studies should examine the rela-
tionship between nutritional deficiency of hamsters in croplands due to intensified mono-
culture and their reproduction performance. Second, the roles of milk production and qual-
ity in offspring hamsters are still unknown, which would obscure the effects of malnutrition
and gut microbes on offspring of hamsters. Therefore, their distinct impacts need further
investigation. Third, the transfer of gut microbes between mother hamsters and their off-
springwasnotassessed,whichpreventsusfromevaluatingtheeffectsofmaternalgut
microbiota on offspring microbiota. Besides, the impacts of caging the animal or rodent
chow on microbial input to offspring also need to be assessed. Finally, microbiota transplan-
tation experiments and multiomic analysis are necessary to identify malnutrition-induced
gut microbiome disturbances and to examine their potential impacts on physiological and
reproductive performance of hamsters.
Conclusion. Our results demonstrate that maternal protein deficiency has a more
profound negative effect on fitness and the gut microbes of hamsters than maternal
niacin deficiency, and such an effect is more pronounced in offspring than in female
hamsters. Our study suggests that malnutrition and associated gut microbes may be a
potential factor in causing the population decline of the greater long-tailed hamster in
the farmland of the North China Plain due to intensified monoculture. Conservation
practices should therefore consider surveying the dietary species richness and host-
associated microbiota of animals (60), which may help explain the bottom-up effects
of human disturbance on their populations. Future studies should emphasize ways to
overcome the above limitations of the study, so as to reveal the roles of gut microbes
in affecting the fitness of greater long-tailed hamsters due to nutritional deficiency
caused by intensified monoculture in farmlands.
MATERIALS AND METHODS
Experimental design. The greater long-tailed hamsters (T. triton) used in this study were obtained
from a laboratory breeding colony at the Institute of Zoology, Chinese Academy of Sciences (CAS). A
total of 102 sexually naive hamsters (51 of each sex, 6 to 7 months of age) from different litters were
used in this study. T. triton is a solitary, polygamous rodent species with intense aggressive behavior dur-
ing conspecific encounters. Individuals were separately housed in polypropylene cages that contained
corncob fragments as bedding for 1 month ahead of the experiment and had free access to food
(AIN93G rodent diet, Beijing KeAo Bioscience Co.) and water. All cages were maintained under a 16-h
light/8-h dark cycle, and the temperature in the animal room was maintained at 22 62°C. We designed
a 2- by 2-factor experiment to test the effects of maternal protein and niacin deficiency on female ham-
sters and their offspring (Table 2). The diet formula was modified based on standard AIN93G rodent
chow. Female (maternal) hamsters were randomly allocated to four diet treatment groups with combi-
nation of low-protein diet (LPD) or normal-protein diet (NPD) and niacin supplement (niacin
1
) or niacin
deficiency (niacin2): (i) normal-protein and niacin-supplemented group, (ii) low-protein and niacin-sup-
plemented group, (iii) normal-protein and niacin-deficient group, and (iv) low-protein and niacin-defi-
cient group (for sample size, see Table 2). Adult males used for mating with maternal females were fed
with standard AIN93G rodent chow during the study (Fig. 1C).
Adult female hamsters (n= 51) were fed with the above modified rodent chow for 1 month before
the breeding experiment (Fig. 1). Body weight of the adult female hamsters was measured at days 0, 6,
10, 14, 18, 22, and 30 to represent the body growth condition of female hamsters. As T. triton displays
very intense aggressive behavior toward conspecifics or people, to minimize handling stress for ham-
sters, the length of body or tail was not measured as the body growth indicator in this study. After a 30-
day experimental period, fecal samples of female hamsters were collected for 2 days to assess the effects
of different diets on the maternal female’s gut microbiota (Fig. 1A). We used an additional neutral arena
box, following a previous study (61), to conduct the cohabitation tests to measure the reproduction per-
formance of female hamsters (Fig. 1B). The box was divided into two cells with a movable partition (see
the black plate in Fig. 1B); each cell contains a fixed protective screen (see the small gray plate in Fig. 1B)
as a shelter to avoid intense biting between hamsters. The cohabitation box was cleaned using water
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
November/December 2022 Volume 10 Issue 6 10.1128/spectrum.00157-22 11
and 75% ethanol between cohabitation tests to eliminate odor and residue. The experiment was con-
ducted in the first 4 h at the beginning of the dark cycle. An adult female (with an opened, moist, and
pinkish vagina) and a randomly selected male were placed in the two cells of the box (80 by 80 by
100 cm) for a 5-min acclimation period, and then the middle partition of the box was removed to start
the cohabitation test. Once the hamster pair in the cohabitation box displayed mating behavior (mount-
ing, thrusting, and intromission) more than three times in 15 min, the female was no longer assigned to
mate with other males and housed individually to facilitate pregnancy and giving birth. If the female was
not able to successfully mate with the male, then another male was introduced into the cohabitated box the
next day or when another estrous cycle for the female began. If no mating behavior of the hamster pair was
observed in 14 days, or the female was injured during the cohabitation test, she was excluded from further
analysis. In total, nine females died or were injured during the study and 19 females gave birth (although one
case of infanticide was observed under the NPD-niacin
2
group [see Table S1 in the supplemental material]).
The pregnancy period of a hamster lasted 26 to 28 days, with litter sizes ranging from 1 to 6. The lactation pe-
riod of litters lasted 26 to 27 days. Body weights of the total 68 pups from four treatment groups (5, 5, 5, and
3 litters from normal-protein and niacin
1
, normal-protein and niacin
2
,low-proteinandniacin
1
,andlow-pro-
tein and niacin
2
groups, respectively) were sampled to represent the body weights of offspring hamsters.
Mating behaviors, including mount latency, mount frequency, number of cohabitation tests, and litter
size were recorded to represent the reproduction performance of female hamsters. Mount latency was
defined as the time needed for a female to have the first copulation with a male that displayed mounting,
thrusting, and intromission while the female showed a typical lordosis posture. Mount frequency was defined
as the number of copulations of the hamster pair within 15 min. The cohabitation test ended when the chas-
ing behavior of male individuals was not observed or the female started to attack the male after several cop-
ulation attempts. Litter size was defined as the number of pups of a female hamster at birth. The number of
cohabitation tests was defined as the number of days required for a female to successfully mate with a male.
Higher mount latency and number of cohabitation tests, lower mount frequency, and smaller litter size indi-
cate a reduction of reproduction performance in a female hamster.
Fecal sample collection. Fecal samples from the female hamsters were collected within 2 days before
cohabitation tests, while offsprings’fecal samples were collected immediately (within 1 to 2 days) when they
were separated from the female hamsters at weaning (Fig. 1A). Hamsters were moved to a clean empty cage
with a bottom of stainless-steel mesh in order to collect fecal samples. Fecal boli were collected immediately
andstoredat280°C for DNA extraction. In total, 36 fecal samples from 45 female hamsters and 68 fecal sam-
ples from 68 offspring hamsters at weaning were collected, stored, and used for further analysis. One litter in
the niacin deficiency group was removed from amplicon analysis because of maternal infanticide (Table S1).
The animal raising and handling were in line with guidance by the Animal Care and Use Committee of
Institute of Zoology, Chinese Academy of Sciences. The authors that conducted the animal experiments were
trained by the Beijing Agency for Experimental Animals, China, with an authorized diploma.
Amplicon sequencing and analysis. The total DNA of the fecal samples was extracted using a
NucleoSpin 96 Soil kit (Macherey-Nagel, Germany) based on the manufacturer’s protocol. DNA concentration
was measured by fluorometry using the Qubit double-stranded DNA (dsDNA) assay kit and fluorometer (Life
Technologies, Carlsbad, CA, USA). The V3-V4 region of the 16S rRNA gene was amplified for 20 cycles using
universal primers (forward primer 338F, 59-ACTCCTACGGGAGGCAGCA-39; reverse primer 806R, 59-GGACT
ACHVGGGTWTCTAAT-39) with the primers containing adapter and barcode sequences. PCR amplification was
performed with the following thermocycling conditions: an initial denaturation at 95°C for 5 min, followed by
15 cycles at 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 7 min. The PCR
products from the first PCR step were purified using VAHTS DNA Clean Beads, and a second round of PCR was
performed in a 40-
m
L reaction mixture containing 20
m
L2Ph
m
sion HS (high-fidelity) master mix, 8
m
Ldou-
ble-distilled water (ddH
2
O), 10
m
M each primer, and 10
m
L of PCR products from the first step. The thermocy-
cling conditions for the second round of PCR were as follows: an initial denaturation at 98°C for 30 s, followed
by 10 cycles at 98°C for 10 s, 65°C for 30s, and 72°C for 30 s, with a final extension at 72°C for 5 min. Finally, all
PCR products were quantified using Quant-iT double-stranded DNA (dsDNA) HS (high-sensitivity) reagent and
were pooled. High-throughput sequencing of bacterial rRNA genes was performed with the purified, pooled
sample using an Illumina HiSeq 2500 platform (2 250 paired ends) at Biomarker Technologies Corporation,
Beijing, China. Sterile water was used as the negative control in DNA extraction and PCR amplification.
Bioinformatic analysis was conducted with QIIME2 (Quantitative Insight into Microbial Ecology, version
2022-2) (62) software. Raw data FASTQ files were transformed into a format that could be read by the QIIME2
system using the qiime tools import program. Demultiplexed sequences from each sample were quality fil-
tered and trimmed (left at 19 bp and right at 20 bp based on primer length), denoised, and merged using
the DADA2 plugin (63). Any contaminating mitochondrial and chloroplast sequences were filtered using the
QIIME2 feature-table plugin. The feature table of amplicon sequence variants (ASVs) was obtained by qiime
tool export function with table.qza generated by the DADA2 plugin. Representative sequences were obtained
by the feature-table plugin. A total of 5,792 ASVs were kept for further analysis. Taxonomy was assigned using
the naive Bayes feature classifier trained against the Greengenes 13_8 database (64). Diversity metrics were
calculated using the qiime diversity alpha plugin without rarefaction (for feature counts, an average of 22,220,
minimum of 9,963, and maximum of 45,253); sequence depth was included as a model covariate in the anal-
ysis.
a
diversity indices, including Shannon diversity, observed features (the number of observed features for
each sample), Chao1 richness estimator, and Faith’s phylogenetic diversity index were calculated to estimate
the microbial diversity within an individual sample. Unless specified above, the parameters used in the analy-
sis were set as default.
Statistical analysis. (i) Analysis of body weight and reproductive behaviors. Effects of protein
and niacin diets in the 2- by 2-factor design on body weight change of female hamster were analyzed
Nutritional Deficiency Affects Fitness of Hamsters Microbiology Spectrum
November/December 2022 Volume 10 Issue 6 10.1128/spectrum.00157-22 12
by linear mixed model (LMM) in R (65). We used body weight as the response variable, maternal diet
treatment and sampling time as the fixed effects, and hamster identity as the random effect. Differences
in body weights of female hamsters between diet groups (different protein and niacin levels) at the
same sampling time were tested using aov (analysis of variance). We also performed repeatability esti-
mation of body weight data using a mixed-effects model by the rpt function in package rptR (66, 67);
the estimate of weight measurement was shown to be highly repeatable (repeatability = 0.869; 95%
confidence interval [CI], 0.804, 0.908). Normality of body weight was tested using the Shapiro-Wilk test
(P= 0.02). Differences in offsprings’body weights between diet groups were tested using the aov func-
tion after normality test (Shapiro-Wilk test, P.0.05).
Effects of protein and niacin diets and their interaction on mount latency, mount frequency, and
number of cohabitation tests between diet groups were analyzed using the generalized linear model
(GLM) with Poisson distribution probability (link function, log). GLM with binomial distribution (link func-
tion, logit) was performed to test the difference of sex ratio of offspring between different diet groups.
The Anova function in the package car was used to test the significance of variables in GLMs. R
2
values
of GLMs were calculated with the rsq package in R.
(ii) Analysis of aand bdiversities. Differences in
a
diversity, including Shannon diversity, Chao1 index,
observed features (estimates of total features), and phylogenetic diversity between different diet groups,
were tested using a two-way analysis of covariance (ANCOVA) after a normality test (Shapiro-Wilk test, P.
0.05) in R; sampling depth was included as a covariate to control for differences in library size.
Analysis of compositional data referred to a guide described by Quinn et al. (68). A Bayesian multipli-
cative replacement strategy was conducted to handle the zero counts in the feature table by package
zCompositions (1.4.0-1) (69) in R, and then the feature tables were subjected to clr (central log ratio)-
transformation by the composition (v.2.0-4) package. Euclidean distance matrices were computed from
transformed ASV table by the vegan package. Analysis of multivariate homogeneity of group dispersions
was conducted in the vegan package (70) in R, while permutational multivariate analysis of variance
(PERMANOVA) of the mixed-effects model based on Euclidean distance matrices was performed with
PRIMER 7 software (71, 72) (with 999 permutations of residuals under a reduced model and a type I sum
of squares). Protein and niacin levels were treated as fixed factors, while the dam’s ID was treated as ran-
dom factor in the model. Principle-coordinate analyses (PCoAs) were also performed to visualize the dis-
similarity of gut microbial composition between samples based on Euclidean distance matrices.
(iii) Analysis of differential abundance at genus level. TheANCOMBC(analysisofcompositionsof
microbiomes with bias correction) algorithm controlled the false-discovery rate (FDR) better than other meth-
ods (73). Differential abundance analysis of genus taxa was performed using ANCOM-BC methodology in R
package ANCOMBC (73). The taxonomy table, feature table, phylogenetic tree file, and metadata table were
merged into a phyloseq object by the phylose q package (74) and then used for the ANCOMBC algorithm.
Taxa with proportions of zeroes greater than 0.9 in all samples were dropped from the analysis. Pvalues
were adjusted with the Holm procedure, and the cutoff threshold of the adjusted Pvalue was 0.05.
(iv) Microbial functional profiling. To further investigate the effects of protein and niacin deficiency
on functional repertoires of gut microbes, we used the ASV unique sequences and feature table to infer func-
tion orthologs and KEGG pathway profiles. The relative abundance of predicted functions and pathway of
each sample were gained using the Tax4fun2 package(75)withdefaultsettingsinR.TheBetadisper function
in the vegan package was used to analyze multivariate homogeneity of group dispersions. Differences in pro-
files of KO functional orthologs and KEGG pathways were computed with PRIMER7 software. PCoA was per-
formed here to depict sample dispersions and variations between dietary treatment groups. PERMANOVA
and PCoA were both conducted using the Bray-Curtis dissimilarity matrix. Downstream differential analysis of
KEGG functions and pathways was conducted in LEfSe (76) with default parameters. (The threshold for the
LDA score was 2.0, and Pvalues were adjusted with the Bonferroni procedure).
Data availability. Raw sequence data can be acquired from the NCBI Sequence Read Archive (SRA)
under BioProject accession no. PRJNA816668.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.4 MB.
ACKNOWLEDGMENTS
This study was supported by the Strategic Priority Research Program of Chinese
Academy of Sciences (grant no. XDPB16, XDB11050300), the International Partnership
Program of Chinese Academy of Sciences (grant no. 152111KYSB20160089), and the
External Cooperation Program of BIC, Chinese Academy of Sciences (grant no.
152111KYSB20150023).
We declare no conflict of interest.
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