Abstract
Simple Summary
Zinc is a vital nutrient required by all living organisms; however, its
impact varies based on Zn concentration and chemical form. This study
examined the effect of zinc chloride (ZnCl[2]) and zinc sulfate
(ZnSO[4]) on the life history performance and hemolymph metabolism of
the common moth, Spodoptera litura, which is known to damage many
crops. We found that, while low levels of ZnCl[2] benefit the
reproduction of Spodoptera litura, higher levels of ZnCl[2] prolong the
preadult developmental period and decrease the preadult survival rate.
Additionally, dietary ZnSO[4] exerts a devastating effect on the
survival of S. litura larvae, even at the lowest concentration. This
helps us better understand the effect of the chemical forms and
concentrations of zinc on the biological processes and toxicological
impacts on insects.
Abstract
Zinc is an essential micronutrient crucial in various biological
processes of an organism. However, the effects of zinc vary depending
on its chemical form. Therefore, the aim of this study was to conduct a
comparative analysis of the life history performances and hemolymph
metabolism of Spodoptera litura exposed to different concentrations of
dietary zinc chloride (ZnCl[2]) and zinc sulfate (ZnSO[4]), utilizing
two-sex life tables and untargeted metabolomics. The preadult survival
rate of S. litura significantly decreased, while the preadult
developmental period of S. litura was prolonged as the dietary ZnCl[2]
concentration increased. However, the fecundity of S. litura at 50
mg/kg dietary ZnCl[2] was significantly increased. The intrinsic rate
of increase (r) and the finite rate of increase (λ) in S. litura in the
control group (CK, no exogenous ZnCl[2] or ZnSO[4] added) and with 50
mg/kg dietary ZnCl[2] were significantly higher than those at 100
mg/kg, 200 mg/kg, and 300 mg/kg. Dietary ZnSO[4] exerts a devastating
effect on the survival of S. litura. Even at the lowest concentration
of 50 mg/kg dietary ZnSO[4], only 1% of S. litura could complete the
entire life cycle. Furthermore, as the dietary ZnSO[4] concentration
increased, the developmental stage achievable by the S. litura larvae
declined. High-throughput untargeted metabolomics demonstrated that
both 100 mg/kg dietary ZnCl[2] and ZnSO[4] decreased the hemolymph
vitamins levels and increased the vitamin C content, thereby helping S.
litura larvae to counteract the stress induced by ZnCl[2] and ZnSO[4].
Simultaneously, dietary ZnCl[2] obstructed the chitin synthesis pathway
in the hemolymph of S. litura, thus extending the developmental period
of S. litura larvae. These results indicate that low concentrations of
Zn^2+ positively impact populations of S. litura, but the effectiveness
and toxicity of Zn depend on its chemical form and concentration.
Keywords: two-sex life table, Spodoptera litura, untargeted
metabolomics, heavy metal stress
1. Introduction
Zinc, an essential micronutrient metal, is crucial for signal
transduction, DNA replication, transcription, and protein synthesis in
organisms, where it performs structural, catalytic, and regulatory
functions [[32]1,[33]2,[34]3]. Additionally, zinc competes with heavy
metal ions, such as cadmium, for transporter-mediated ion transport;
this competition reduces the cellular absorption rate of heavy metal
ions, thereby shielding cells from their toxic effects
[[35]4,[36]5,[37]6]. Since the 1930s, zinc has been widely acknowledged
as a crucial component in the growth processes of both animals and
plants; it is routinely added to animal feeds and plant fertilizers as
a nutritional supplement to enhance healthy growth and optimize
production [[38]7,[39]8]. However, when zinc concentrations surpass an
organism’s regulatory capacity, it damages the midgut structure
[[40]9], precipitating neuronal apoptosis or necrosis [[41]10], as well
as physiological, behavioral, and metabolic dysfunctions
[[42]11,[43]12], ultimately diminishing population survival and
fecundity [[44]13,[45]14].
Zinc pollution has emerged as a widespread issue globally
[[46]15,[47]16,[48]17]. Zhuang et al. (2009) observed that the average
zinc concentration in the surface soil of farmland in the Pearl River
Delta significantly exceeded the Environmental Quality Standard of
soils in China [[49]18]. Similarly, Wu et al. (1996) reported that zinc
concentrations in the leaves and stems of cabbage grown in
zinc-enriched soil reached values as high as 1297 and 1141.8 mg/kg,
respectively [[50]19]. Additionally, zinc deposition in agricultural
soil through organic fertilizers (livestock manure) is a significant
factor contributing to zinc enrichment in both the soil surface and
plants [[51]15,[52]20]. Researchers have demonstrated that the zinc
levels in organic waste (pig and cow manure) progressively increase
during composting, resulting in zinc concentrations that significantly
exceed those of other heavy metals such as lead (Pb), copper (Cu), and
nickel (Ni) in the Netherlands, England, Wales, and China
[[53]21,[54]22,[55]23,[56]24,[57]25]. In Japan, zinc has been
classified as a pollutant of concern and designated as a model toxicant
due to its environmental and health impacts [[58]26].
Zinc chloride (ZnCl[2]) and zinc sulfate (ZnSO[4]) are prevalent zinc
salts in both daily life and industry [[59]27]. ZnCl[2], a compound
rarely found in nature, is predominantly synthesized industrially.
Conversely, ZnSO[4] is found in a variety of minerals and serves as the
primary zinc additive in feeds and fertilizers [[60]28]. The global
market for ZnSO[4], driven by increasing demand in sectors such as
agriculture, animal husbandry, industry, food, and pharmaceuticals, has
an annual growth rate of 4.5%
([61]https://dataintelo.com/report/zinc-sulfate-market/, accessed on 14
July 2024). Although the market demand and usage of ZnSO[4] surpass
those of ZnCl[2], researchers predominantly utilize ZnCl[2] as a zinc
source to assess the effects of excessive zinc intake on insect growth,
development, reproduction, physiological functions, immune responses,
enzymatic reactions, and metabolism
[[62]9,[63]12,[64]29,[65]30,[66]31]. Researchers, however, more
frequently study the inhibitory effects of high-concentration ZnSO[4]
solutions, which are used as insecticides sprayed on plant leaves, on
the growth, development, and survival of herbivores [[67]32,[68]33].
Therefore, given the widespread use of zinc sulfate in agriculture,
animal husbandry, industry, and food production, ZnSO[4] should be
considered as a dietary source to assess the impact of zinc pollution
on insects.
Spodoptera litura (Fabricisu, 1775) (Lepidoptera, Noctuidae), an
important polyphagous agricultural pest globally, damages cotton,
tobacco, soybeans, corn, and cabbage, among other crops, affecting more
than 90 crops [[69]34]. Due to its well-defined artificial diet and
ease of laboratory handling, S. litura has become an important model
organism for researchers studying the effects of heavy metals on insect
life history performances, metabolism, ion transport, and more
[[70]35,[71]36]. High-throughput untargeted metabolomics is used to
detect differential metabolites of organisms under different
treatments, thereby elucidating their metabolic processes
[[72]37,[73]38,[74]39]. In this study, we aimed to accurately evaluate
the effects of 0 mg/kg (CK), 50 mg/kg, 100 mg/kg, 200 mg/kg, and 300
mg/kg dietary ZnSO[4] and ZnCl[2] on the growth, development,
reproduction, and population dynamics of S. litura using a two-sex life
table. Concurrently, high-throughput untargeted metabolomics based on
liquid chromatography–mass spectrometry (LC-MS/MS) was employed to
reveal the regulation of metabolites in the hemolymph of S. litura in
response to dietary ZnCl[2] and ZnSO[4]. The results will provide a
deeper understanding for the toxic effects of Zn^2+ and SO[4]^2- and
offer new insights for the population management of S. litura.
2. Materials and Methods
2.1. Artificial Diet Content and Preparation
The artificial diet weighed 139 g, consisting of 71.94% pure water (100
g) and 28.06% dry matter (39 g). The artificial diet contained the
following: soybean flour, corn flour, and wheat germ, each at 10 g;
yeast extract powder at 1.2 g; sucrose at 5 g; agar at 1.6 g; vitamin C
at 0.6 g; and sorbic acid, multi-vitamin, methyl p-hydroxybenzoate, and
additional sorbic acid, each at 0.2 g. According to Jin et al., 2020,
the ZnCl[2] and ZnSO[4] (Tianjin Guangfu Fine Chemical Research
Institute, China) content added to the artificial diets was calculated
using the following formula [[75]29]:
weight of ZnCl[2] = Zn concentration of diets × wet weight of diets ×
136.315/65.39, (1)
weight of ZnSO[4] = Zn concentration of diets × wet weight of diets ×
161/65.39, (2)
where 136.315, 161, and 65.39 represent the molecular weights of
ZnCl[2], ZnSO[4], and Zn, respectively. Therefore, in the artificial
diet, the 50, 100, 200, and 300 mg/kg Zn^2+ concentrations correspond
to 14.49 mg, 28.98 mg, 57.96 mg, and 86.94 mg of ZnCl[2], respectively,
and to 17.11, 34.22, 68.44, and 102.66 mg of ZnSO[4], respectively.
Following the methods of Zhang et al. (2016) [[76]40] and Yang et al.
(2022) [[77]36], agar was added to boiling pure water (100 mL), and the
mixture was continuously heated and stirred until the agar completely
dissolved. Subsequently, different amounts of ZnCl[2] and ZnSO[4] were
dissolved in the solution prior to adding the mixture of soybean flour,
corn flour, wheat germ, sucrose, and yeast. The mixture was then
continuously heated and stirred for approximately 10 min. Finally, the
artificial diet was poured into transparent plastic boxes measuring
15.5 × 11 × 6 cm, after the temperature of the mixture dropped below 60
°C.
2.2. Insects Source and Rearing
S. litura were obtained from the College of Life Sciences, Sun Yat-sen
University (Guangzhou, China), in September 2018 and fed a standard
artificial diet for more than 10 generations. S. litura were maintained
under constant conditions: 25.0 ± 0.5 °C, 50 ± 10% relative humidity,
and a 12 h dark/light photoperiod. A cohort of 100 newly hatched
larvae, randomly selected from egg masses laid on the same day, were
individually housed in plastic Petri dishes (4 cm diameter, 1 cm
height) and fed artificial diets containing varying concentrations of
ZnCl[2] and ZnSO[4]. The survival and molting of S. litura larvae were
recorded daily to track the developmental stage. The black exoskeleton
shed by the S. litura specimens indicated that they had developed to
the next instar. Additionally, if the S. litura larvae did not move
when poked with a brush, did not curl up, appeared soft, and their body
color became darker, this indicated that they were dead. Upon reaching
the prepupal stage, each larva was relocated to an individual
compartment in a 32-compartment tray containing sterile sand to
facilitate pupation. The key characteristics of the prepupal stage in
S. litura include a shorter and broader body, the cessation of feeding,
dark brown heads, and a darkened, shiny dorsum. Additionally,
prepupal-stage S. litura larvae secrete mucus to begin constructing a
pupal chamber. Following eclosion, male and female moths that developed
on the same dietary treatment and emerged simultaneously were paired in
plastic cups (4.3 × 6.7 × 7.5 cm) with wax paper to enable oviposition.
If the number of emergent females exceeded that of males, or vice
versa, additional specimens were supplemented from a mass-reared
population maintained under identical conditions to ensure that all S.
litura adults were mated. Nutrition for the adult moths was provided by
a cotton ball soaked in a 10% honey–water solution, which was renewed
daily at the base of each cup. Egg masses deposited on the wax paper
were collected daily, and individual eggs were counted using a stereo
microscope (SMZ745, Nikon, Tokyo, Japan) until the expiration of all
female adults. The longevity of all S. litura adults under different
treatments was recorded.
2.3. Life Table Analysis
The life history performances (larval and pupal developmental times,
survival rates, adult longevity, and fecundity) and population
parameters (intrinsic rate of increase r, finite rate of increase λ,
net reproductive rate R[0], and mean generation time T) of S. litura
were based on the two-sex life table theory, as described by Chi and
Liu (1985) [[78]41] and Chi (1988) [[79]42], and they were calculated
using the TWOSEX-MSChart software Version 2024.07.06 [[80]43]. The
bootstrap method with 100,000 resamples, as proposed by Efron and
Tibshirani (1993) [[81]44], was utilized to determine the means and
standard errors of these parameters. In addition, the bootstrap
technique was also employed to compare the significant differences in
S. litura’s life history parameters and population parameters between
the different groups. This study further explored the age–stage
survival rate (s[xj]), which is the probability that a newborn (age 0,
stage 1) survives to age x and stage j [[82]45]. The age-specific
survival rate (l[x]), representing the probability that a newborn
survives to age x, was also analyzed alongside its relationship with
s[xj]:
[MATH: lx=∑j=1ksx<
/mi>j,
:MATH]
(3)
where k is the number of stages. The age-specific fecundity (f[x]) is
defined as the number of eggs produced by adult females at age x. The
net reproductive rate (R[0]) is a key demographic parameter
representing the average number of offspring an individual is expected
to produce over its lifetime.
[MATH: R0=∑x=0∞lxmx
:MATH]
(4)
The intrinsic rate of increase (r), indicative of the maximum transient
growth rate which a stable age-structured population can achieve, is
calculated employing the iterative bisection method based on the
Euler–Lotka equation, as described by Goodman (1982) [[83]46]:
[MATH: ∑x=0∞e−<
/mo>r(x+1)lxmx=1 :MATH]
(5)
The finite rate of increase (λ), which measures the daily average rate
at which a population grows when not limited by environmental
constraints, is calculated using the following expression:
λ = e^r (6)
The mean generation time (T) is defined as the time required for a
population to increase to R[0]-fold of its size under a stable
age–stage distribution. This parameter quantifies the average period
necessary for a population to replicate its size according to the net
reproductive rate.
T = (ln R[0])/r (7)
2.4. Extraction and Pretreatment of Metabolite
After completing the experiment on the effects of various
concentrations of dietary zinc chloride and zinc sulfate on the life
history parameters of S. litura, we began extracting metabolites from
the hemolymph of S. litura larvae in August 2023. Sixth instar larvae
of S. litura, fed on diets with CK, 100 mg/kg dietary ZnCl[2], and 100
mg/kg dietary ZnSO[4], were first cleansed using deionized water and
subsequently disinfected with 75% alcohol on their body walls. These
larvae were then temporarily immobilized by exposure to −10 °C for 10
min. Using a sterile insect needle, the larval body wall was punctured
at the mid-abdomen, and hemolymph was collected using microcapillaries.
The extracted hemolymph was transferred into 1.5 mL centrifuge tubes
pretreated with 0.025% phenylthiocarbamide to prevent blackening
reactions and then stored at −80 °C. For each experimental treatment,
hemolymph was collected from 80 larvae, and 1 mL of hemolymph was
pooled from every 10 larvae to form a sample, with each treatment
replicated eight times.
Metabolite pretreatment of the S. litura hemolymph for ultraperformance
liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) Agilent
Technologies Inc., California, and SUA, analysis was conducted
following the protocol described by Cheng (2023) [[84]47]. Briefly, 200
μL of thawed hemolymph was mixed with 800 μL of methanol and agitated
for 60 s. This mixture was then centrifuged at 14,000 rpm for 10 min at
4 °C. The resulting supernatant was transferred to a new centrifuge
tube and subjected to freeze-drying. The dry sample was reconstituted
in 400 µL of a 4 ppm 2-chlorophenylalanine-methanol aqueous solution
(1:1, v/v) at 4 °C and filtered through a 0.22 µm filter membrane into
a sampling vial for subsequent HPLC-MS/MS analysis. Additionally, 20 μL
from each prepared sample was pooled to create a quality control (QC)
sample, used to correct deviations in analytical results and
instrumentation errors.
2.5. UHPLC-MS/MS Analysis
Metabolites in the hemolymph of S. litura were characterized and
quantified using UHPLC-MS/MS on a Waters ACQUITY UPLC HSS T3 system.
Chromatography was performed using an ACQUITY UPLC HSS T3 column
Beijing Yuwei Technology Co., Ltd, Beijing, and China, (150 × 2.1 mm,
1.8 μm, Waters) with the autosampler temperature maintained at 8 °C and
the column temperature at 40 °C. An injection volume of 2 μL was used.
The mobile phases, composed of formic acid in water (1:1000, v/v) and
formic acid in acetonitrile (1:1000, v/v), were delivered at a flow
rate of 0.25 mL/min. Mass spectrometry was performed using electrospray
ionization in both positive (ESI+) and negative ion modes (ESI−) with
capillary temperatures set at 325 °C and spray voltages of 3.8 kV
(positive) and −2.5 kV (negative). The mass analyzer operated from 81
to 1000 m/z at a resolution of 70,000 in full scan mode.
Peak area data for hemolymph metabolites from larvae fed diets with CK,
100 mg/kg ZnCl[2], and 100 mg/kg ZnSO[4] concentrations were
normalized. A total of 501 metabolites annotated in the hemolymph of S.
litura larvae were used for principal component analysis (PCA) and
partial least squares discriminant analysis (PLS-DA). In SIMCA-P v.
14.0, PCA and PLS-DA were employed for unsupervised and supervised data
analysis, respectively, aiding in data clustering and preventing model
overfitting. Metabolites demonstrating a Variable Importance in the
Projection (VIP) > 1, a p-value < 0.05 (t-test), and a Fold Change (FC)
> 1.2 or <0.833 were identified as significantly different across the
treatments. Metabolite annotation was conducted using the KEGG database
([85]https://www.genome.jp/kegg/, accessed on 24 July 2024), which
provided insights into the biochemical metabolic and signal
transduction pathways of the identified differential metabolites.
3. Results
3.1. Effects of Dietary ZnCl[2] on the Development and Survival of S. litura
The developmental period of the third instar larvae, fourth instar
larvae, fifth instar larvae, and preadults of S. litura was
significantly extended as the concentration of ZnCl[2] increased.
Compared to the preadult developmental period of S. litura at 0 mg/kg
dietary ZnCl[2] (48.5 d), the preadult developmental period of S.
litura at 100 (57.8 d), 200 mg/kg (59.4 d), and 300 mg/kg (56.4 d)
dietary ZnCl[2] was significantly extended by 9.3 d, 10.9 d, and 7.9 d,
respectively. Simultaneously, the preadult survival rate of S. litura
significantly decreased as the dietary ZnCl[2] concentration increased
([86]Table 1). Compared to a preadult survival rate of S. litura at 0
mg/kg ZnCl[2] (70.0%), the preadult survival rates at 50 mg/kg (47.9%),
100 mg/kg (33.0%), 200 mg/kg (27.0%), and 300 mg/kg (5.4%) dietary
ZnCl[2] were significantly decreased by 22.1%, 37.0%, 43.0%, and 64.6%,
respectively ([87]Table 1). The age–stage survival rates (s[xj]) and
age-specific survival rate (l[x]) for S. litura pupae decreased sharply
at dietary ZnCl[2] concentrations of 50 and 100 mg/kg, whereas, for
stages L6–8, the age–stage survival rates (s[xj]) and age-specific
survival rate (l[x]) declined rapidly at 200 and 300 mg/kg ([88]Figure
1A–E and [89]Figure 2A).
Table 1.
The life history parameters of S. litura on diets with different
ZnCl[2]concentrations.
Life History Parameters Concentration (mg/kg)
0 50 100 200 300
Egg (d) 3.0 ± 0.00a (100) 3.0 ± 0.00a (100) 3.0 ± 0.00a (100) 3.0 ±
0.00a (100) 3.0 ± 0.00a (100)
L1 (d) 5.0 ± 0.17a (99) 4.8 ± 0.15a (88) 4.8 ± 0.15a (100) 4.7 ± 0.12a
(100) 4.7 ± 0.14a (100)
L2 (d) 3.9 ± 0.07a (88) 3.4 ± 0.09bc (83) 3.2 ± 0.08c (100) 3.5 ± 0.09b
(100) 3.5 ± 0.13bc (100)
L3 (d) 3.1 ± 0.06d (84) 3.2 ± 0.10cd (80) 3.3 ± 0.08c (94) 3.6 ± 0.10b
(100) 4.2 ± 0.18a (91)
L4 (d) 3.2 ± 0.05d (84) 3.3 ± 0.09d (77) 3.5 ± 0.05c (87) 4.1 ± 0.08b
(97) 4.5 ± 0.12a (84)
L5 (d) 3.4 ± 0.06c (81) 3.5 ± 0.06c (68) 4.2 ± 0.06b (83) 5.4 ± 0.15a
(86) 5.5 ± 0.24a (61)
L6–8 (d) 12.6 ± 0.22d (81) 14.4 ± 0.34c (66) 24.4 ± 0.82a (77) 23.4 ±
0.80a (42) 19.1 ± 1.17b (19)
Prepupa 1.45 ± 0.07c (81) 1.51 ± 0.06bc (66) 1.92 ± 0. 05a (77) 1.95 ±
0.07a (42) 2.01 ± 0.16a (19)
Pupa (d) 13.6 ± 0.21a (76) 13.1 ± 0.13ab (48) 13.0 ± 0.12b (33) 13.0 ±
0.14b (27) 13.4 ± 0.20ab (5)
Preadult (d) 48.5 ± 0.44c (66) 48.8 ± 0.51c (48) 57.8 ± 0.54b (33) 59.4
± 0.26a (27) 56.4 ± 0.99b (5)
Preadult survival rate (%) 66.0 ± 4.74a (66) 47.9 ± 4.99b (48) 33.0 ±
4.69cd (33) 27.0 ± 4.43d (27) 5.4 ± 2.04e (5)
Oviposition days (d) 6.5 ± 0.75b (34) 7.3 ± 0.85b (24) 10.7 ± 1.17a
(15) 4.8 ± 1.35b (15) 6.9 ± 3.04 (2)
Female longevity (d) 14.1 ± 1.69a (34) 15.8 ± 1.83a (24) 15.9 ± 2.32a
(15) 12.7 ± 0.92a (15) 12.5 ± 4.93 (2)
Male longevity (d) 20.9 ± 1.92b (32) 17.8 ± 2.11b (24) 23.3 ± 2.58ab
(18) 23.2 ± 3.27ab (12) 31.5 ± 2.94a (3)
Fecundity 620.4 ± 137.30b (34) 1022.4 ± 247.13a (24) 895.3 ± 238.30ab
(15) 592.9 ± 246.71b (15) 900.7 ± 106.67 (2)
[90]Open in a new tab
The means and standard errors were calculated using the bootstrap
procedure with 100,000 resamples. The means followed by different
letters in the same row are significantly different at the 5%
significance level. L1 (first instar); L2 (second instar); L3 (third
instar); L4 (fourth instar), L5 (fifth instar); and L6–8 (sixth instar
to the eighth instar). The sample sizes for oviposition days, female
longevity, and fecundity in the 300 mg/kg Zn group were only N = 2,
indicating an insufficient power to detect meaningful differences.
Therefore, the oviposition days, female longevity, and fecundity in the
300 mg/kg dietary ZnCl[2] group were excluded from the analyses.
Figure 1.
[91]Figure 1
[92]Open in a new tab
Effect of the CK (A), dietary ZnCl[2] (B–E) and ZnSO[4] (F–I) on the
age–stage survival rate (s[xj]) of S. litura.
Figure 2.
[93]Figure 2
[94]Open in a new tab
Effect of dietary ZnCl[2] (A) and ZnSO[4] (B) on the age-specific
survival rate (l[x]) and age-specific fecundity (f[x]) (C) of S.
litura.
3.2. Effects of Dietary ZnCl[2] on Longevity and Fecundity of S. litura
Adults
There was no significant difference in the longevity of S. litura
female adults across different dietary ZnCl[2] concentrations. However,
the longevity of males at 300 mg/kg (31.4 d) was significantly longer
than at 0 mg/kg (20.9 d) and 50 mg/kg (17.8 d) ([95]Table 1). The
fecundity of S. litura females at 50 mg/kg (1022.4 egg/female) dietary
ZnCl[2] was significantly higher than that at 0 mg/kg (620.4
eggs/female) and 200 mg/kg (592.9 eggs/female) ([96]Table 1). The
oviposition days of S. litura females on 100 mg/kg (10.7 d) dietary
ZnCl[2] were significantly longer than those on 0 mg/kg (6.5 d), 50
mg/kg (7.3 d), 200 mg/kg (4.8 d), and 300 mg/kg (6.9 d) dietary
ZnCl[2]. The age-specific female fecundity curves (f[x]) showed two
egg-laying peaks of S. litura female at various ZnCl[2] concentrations
([97]Figure 2). The maximum f[x] value for S. litura at 50 mg/kg (210
egg/female) and 300 mg/kg (305 eggs/female) was higher than at 0 mg/kg
(122.2 eggs/female), 100 mg/kg (172 eggs/female), and 200 mg/kg (157
eggs/female) ([98]Figure 2C).
3.3. Effects of Dietary ZnCl[2] on the Population Parameters of S. litura
The intrinsic rate of increase (r) and the finite rate of increase (λ)
of S. litura significantly decreased with increasing dietary ZnCl[2]
concentrations. There was no significant difference in the r and λ of
S. litura at the CK (r = 0.092, λ = 1.0963) and 50 mg/kg (r = 0.0985, λ
= 1.1035) dietary ZnCl[2] concentrations. However, these values were
significantly higher than those at the 100 (r = 0.0728, λ = 1.0755),
200 (r = 0.0637, λ = 1.0658), and 300 mg/kg (r = 0.0494, λ = 1.0507)
mg/kg dietary ZnCl[2] concentrations. There was no significant
difference in the net reproductive rate (R[0]) of S. litura at the CK
(211.05), 50(245.02), and 100 mg/kg (134.46) dietary ZnCl[2]
concentrations, but it was significantly higher than that at the 300
mg/kg (21.16) dietary ZnCl[2] concentration ([99]Table 2).
Table 2.
Population parameters of S. litura specimens reared on diets with
different ZnCl[2] concentrations.
Concentration (mg/kg) Population Parameters
r (d^−1) λ (d^−1) R[0] (Offspring/Individual) T (d)
0 0.0920 ± 0.0058a 1.0963 ± 0.0063a 211.05 ± 54.74a 57.88 ± 1.30b
50 0.0985 ± 0.0065a 1.1035 ± 0.0071a 245.02 ± 72.82a 55.40 ± 1.19b
100 0.0728 ± 0.0059bc 1.0755 ± 0.0064bc 134.46 ± 47.13a 66.39 ± 1.43a
200 0.0637 ± 0.0094cd 1.0658 ± 0.0100cd 88.99 ± 41.63ab 68.18 ± 1.23a
300 0.0494 ± 0.0095d 1.0507 ± 0.0100d 21.16 ± 11.52b 58.99 ± 1.82b
[100]Open in a new tab
Means in the same row followed by different letters are significantly
different at the 5% significance level. r (intrinsic rate of increase),
λ (finite rate of increase), R[0] (net reproductive rate), and T (mean
generation time).
3.4. Effect of Dietary ZnSO[4] on Life History Performances
Only 1% of S. litura larvae on 50 mg/kg dietary ZnSO[4] completed the
entire life cycle, and those fed higher concentrations of ZnSO[4] were
unable to develop into adults. As the ZnSO[4] concentration increased,
the developmental stages that S. litura larvae reached were
progressively lower ([101]Figure 1F–I). S. litura larvae that were fed
a diet containing 100 mg/kg ZnSO[4] could develop up to the prepupal
stage. However, S. litura larvae on 200 mg/kg dietary ZnSO[4] reached
only L6–8, and those at 300 mg/kg progressed only to L6 ([102]Figure
1H,I). Dietary ZnSO[4] resulted in a sharp drop in the survival rate of
S. litura larvae. Higher concentrations of ZnSO[4] had no significant
effect on the s[xj] of early larval stages (L1, L2, L3, and L4).
Following the onset of the later larval stages, dietary ZnSO[4]
significantly reduced the s[xj] and l[x] of S. litura larvae
([103]Figure 1F–I and [104]Figure 2B).
3.5. Metabolic Profiles of S. litura Fed Diets with Different Treatments
The PCA and PLS-DA models demonstrated complete separation of the CK,
100 mg/kg dietary ZnSO[4], and 100 mg/kg dietary ZnCl[2] at a 95%
confidence level, suggesting significant differences in the metabolic
characteristics between the treatments ([105]Figure 3A,B). Metabolites
showing significant differences were identified from 501 annotated
hemolymph metabolites by setting VIP > 1.0, FC > 1.2 or FC < 0.833, and
p-value < 0.05. In the ZnCl[2] group, 121 metabolites were
significantly upregulated, and 199 were noticeably downregulated
compared to the CK group ([106]Figure 3C). In the ZnSO[4] group, 193
metabolites were substantially upregulated, and 90 were noticeably
downregulated compared to the CK group ([107]Figure 3D). A total of 118
compounds were remarkably upregulated, and 98 metabolites were notably
downregulated in the ZnCl[2] group compared to the ZnSO[4] group
([108]Figure 3E).
Figure 3.
[109]Figure 3
[110]Open in a new tab
Metabolic profile of S. litura reared on diets with CK, 100 mg/kg
ZnCl[2], and 100 mg/kg ZnSO[4]. Principal components analysis, PCA (A);
partial least squares discrimination analysis, PLS-DA (B). The red,
yellow, and green shaded regions surrounding the points represent the
95% confidence interval for the CK, dietary ZnCl[2], and ZnSO[4]
groups, respectively; and volcano plots of changed metabolites of S.
litura on diets with CK vs. ZnCl[2] (C), CK vs. ZnSO[4] (D), and
ZnCl[2]vs. ZnSO[4] (E). The red, blue, and gray dots represent
upregulation, downregulation, and no significant change in the
hemolymph metabolites, respectively. “NoDiff” indicates no significant
difference. Bubble plot of KEGG pathway enrichment analysis (top 9
enriched KEGG pathways) on diets of CK vs. ZnCl[2] (F), CK vs. ZnSO[4]
(G), and ZnCl[2] vs. ZnSO[4] (H).
KEGG pathway enrichment identifies the primary biochemical metabolic
and signal transduction pathways associated with differential
metabolites. In the comparison between CK and ZnCl[2], chitin synthesis
and the vitamin digestion and absorption pathways were identified as
primary biochemical metabolic pathways ([111]Figure 3F), with nine
metabolites annotated for the former, six of which showed significant
differences ([112]Figure 3H), and ten metabolites annotated for the
latter, six of which showed significant differences ([113]Figure 3G).
3.6. Metabolic Pathway of S. litura Fed Diets with Different Treatments
A detailed visual analysis of the metabolic pathways for chitin
synthesis is shown in [114]Figure 4. In chitin synthesis, trehalose is
gradually converted into glucose-6-phosphate, glucosamine 6-phosphate,
N-Acetylglucosamine-6-phosphate, N-Acetyl-glucosamine-phosphate,
UDP-N-Acetylglucosamine-phosphate, and chitin. The relative
concentrations of trehalose, glucose-6-phosphate, glucosamine
6-phosphate, N-Acetylglucosamine-6-phosphate,
N-Acetylglucosamine-phosphate, and UDP-N-Acetylglucosamine-phosphate in
the hemolymph of S. litura larvae fed ZnCl[2] were significantly lower
than those fed CK and ZnSO[4]. The relative content of glutamine in the
hemolymph of S. litura larvae fed ZnCl[2] was significantly higher than
among those fed CK and ZnSO[4] ([115]Figure 4). Additionally, in the
vitamin digestion and absorption metabolic pathway, the relative
content of vitamin H, vitamin B6, vitamin B2, flavin mononucleotide,
and folic acid in the hemolymph decreased significantly in S. litura
fed ZnCl[2] and ZnSO[4], compared to CK. However, the content of
vitamin C in the hemolymph did not show a significant increase
([116]Figure 5).
Figure 4.
[117]Figure 4
[118]Open in a new tab
Chitin synthesis pathway and changes in the relative concentrations of
metabolites. The arrows indicate the direction of the metabolic
pathways. The upper panel shows a series of annotated metabolites in
the process of trehalose being gradually converted into chitin in S.
litura hemolymph. The red borders indicate that the relative
concentration of metabolites in S. litura hemolymph from specimens
reared on dietary ZnCl[2] is significantly higher compared to the CK
and dietary ZnSO[4] treatments. The green borders indicate that the
relative concentration of metabolites in S. litura hemolymph from
specimens reared on dietary ZnCl[2] is significantly lower compared to
the CK and dietary ZnSO[4] treatments. The black borders denote that
these metabolites were not detected by HPLC-MS/MS. The red circles
indicate the relative concentration of the metabolites in each sample.
Statistical analyses were conducted using the t-test (* p < 0.05; ** p
< 0.01).
Figure 5.
[119]Figure 5
[120]Open in a new tab
Vitamin digestion and absorption metabolic pathway and changes in the
relative concentrations of metabolites. The red circles indicate the
relative concentration of the metabolites of each samples. Statistical
analyses were conducted using the t-test (* p < 0.05; ** p < 0.01).
4. Discussion
Zinc, serving as an essential trace nutrient, confers multiple
biological benefits at low concentrations [[121]48]. Additionally, zinc
can shield cells from the toxic effects of various toxins and
contribute to the structural and functional maturation of neurons
[[122]5,[123]35,[124]49,[125]50]. However, an excessive intake of zinc
can prolong the larval development time, reduce the larval survival
rates, and disrupt the metabolic processes in the hemolymph, as well as
physiological and biochemical functions [[126]30,[127]51]. In our
study, the preadult developmental period of S. litura fed on diet with
100, 200, and 300 mg/kg ZnCl[2] concentrations was significantly
extended by 9.3 d, 10.9 d, and 7.9 d, respectively, compared to the CK
group ([128]Table 1). Insects need to expend energy to carry out the
detoxification, storage, or excretion of heavy metals
[[129]31,[130]52,[131]53]. For example, heavy metals may generate an
oxidative stress response, compelling organisms to synthesize
additional peptides and enzymes, such as metallothioneins (MT) and heat
shock proteins (Hsps), to mitigate the toxicity of heavy metals,
resulting in insufficient energy for insects, thereby adversely
affecting their growth, development, and reproduction [[132]54].
Furthermore, excessive zinc binds to the sulfhydryl (-SH) groups in
critical proteins (enzymes involved in various metabolic pathways),
which leads to reduced ATP synthesis and an ensuing energy crisis in
organisms [[133]55]. Consequently, when S. litura consumes excessive
amounts of ZnCl[2] from its diet, it diverts more energy from food
nutrients to detoxification processes, resulting in decreased body
weight and prolonged larval development.
Additionally, the preadult survival rates of S. litura at dietary
ZnCl[2] concentrations of 100, 200, and 300 mg/kg significantly
decreased by 22.1%, 37.0%, 43.0%, and 64.6%, respectively, compared to
the CK group ([134]Table 1). Jin et al. (2020) [[135]29] found that,
when S. litura was fed ZnCl[2] at concentrations ranging from 150 to
450 mg/kg, the relative consumption rate (RCR) and approximate
digestibility (AD) of the sixth instar larvae increased, leading to
rapid zinc accumulation in the body. Moreover, Shu et al. (2012)
[[136]9] observed that the zinc content in the midgut of S. litura
increased with increasing dietary ZnCl[2] concentrations, leading to
the significant formation of metallothioneins (MT) in the midgut and
the emergence of numerous electron-dense granules (EDGs) and vacuoles
in the cytoplasm of midgut cells. This suggests that, during the period
of overeating, S. litura rapidly ingests ZnCl[2] from its diet, failing
to excrete excess zinc, resulting in increased mortality in older
larvae.
Although feeding S. litura high concentrations of ZnCl[2] resulted in a
prolonged preadult developmental period and decreased preadult survival
rates, the fecundity of adults fed 50 mg/kg ZnCl[2] was significantly
higher than that of adults fed the CK diet or higher concentrations
(200 mg/kg) of ZnCl[2] ([137]Table 1). Similarly, Shephard et al.
(2020) [[138]56] found that Lepidopteran pest (Pieris rapae) larvae
developing on intermediate concentrations of ZnCl[2] had higher adult
fecundity. Indeed, zinc, as a major structural component of many
enzymes and transcription factors, can activate regulatory proteins and
increase proliferation and differentiation during vitellogenesis
[[139]57]. Additionally, Falchuk and Montorzi (2001) [[140]58] found
that vitellogenin (Vg) is a metalloprotein containing zinc and calcium.
When Vg is processed into vitellin in the oocyte, zinc is also
transported to the ovary via the hemolymph and taken up by the Vg-bound
oocyte. Therefore, an appropriate intake of zinc can facilitate the
formation of vitellogenin in the eggs of S. litura, thereby enhancing
their reproductive capacity. However, an excessive intake of zinc can
significantly diminish the reproductive capacity of S. litura. Shu et
al. (2009) [[141]30] demonstrated, using Western blotting and
inductively coupled plasma atomic emission spectrometry, that an
excessive zinc intake leads to the accumulation of zinc in the ovaries
of S. litura, thereby disrupting ovarian development, reducing yolk
protein content, and ultimately decreasing fecundity. Similarly,
Al-Dhafar and Sharaby (2012) [[142]32] also observed that zinc
accumulation in yolk granules (vitellogenesis) and follicular
epithelial cells disrupts the production of female gametes, thereby
causing ovarian malformations and reducing the fecundity of insects.
Surprisingly, even when S. litura fed on the highest concentration (300
mg/kg) of ZnCl[2], 5% of the S. litura specimens could complete the
entire life cycle. However, only 1% of the S. litura specimens fed on a
lower concentration (50 mg/kg) of ZnSO[4] could complete the entire
life cycle. Therefore, compared to the effect of ZnCl[2] at the same
concentration on the survival of S. litura, we suspect that dietary
SO[4]^2− might have a devastating effect on the survival of S. litura.
Kavitha et al. (2012) [[143]59] found that dietary ZnSO[4] reduced the
protein levels in the silk gland and hemolymph by 356% and 181%,
respectively. Additionally, sulfate salts, due to their unique
insecticidal properties, also lead to insect mortality. For example,
FeSO[4] is often used as an animal insecticide for the control of stick
insect pests [[144]60,[145]61,[146]62]. However, it is not clear
whether sulfate ions or metal cations cause the death of insects,
although Al-Dhafar and Sharaby (2012) [[147]32] found that a 0.566%
ZnSO[4] solution caused vacuolation and contraction of midgut
epithelial cells and goblet cells in the distal part of the midgut,
contraction of some peritrophic membranes, and shedding of some muscle
layers in Rhynchophorus ferrugineus. Additionally, Halpern et al.
(2002) [[148]63] discovered that CuSO[4] increased the permeability of
the plasma membrane of the midgut cells of Chironomus luridus, allowing
fluid from the intestinal cavity to freely enter the extracellular
space, resulting in death. However, it is difficult for us to determine
whether SO[4]^2− alone or SO[4]^2− and metal cations together
negatively affect insects. From our experimental results, we can
determine that, compared to chloride metal salts, SO[4]^2− has a
devastating effect on the survival of insects, which may be an
important reason why CuSO[4] and FeSO[4] are used as insecticides.
To further explore the regulation of metabolites in the hemolymph of S.
litura under the influence of ZnCl[2] and ZnSO[4] intake, we utilized
untargeted metabolomics to analyze the metabolic characteristics and
profiles. The relative concentrations of glutamine and various
chemicals in the chitin synthesis pathway (Glucose-6-phosphate,
Glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate,
N-acetylglucosamine-1-phosphate, and UDP-N-acetylglucosamine phosphate)
in S. litura on dietary ZnCl[2] were significantly lower than those on
CK and dietary ZnSO[4] ([149]Figure 4). Chitin, a well-known linear
homopolymer composed of β-1,4-linked N-acetylglucosamine, is found in
the anterior cuticle, trachea, and muscle attachment sites, where it
functions alongside proteins and other components to provide structural
support for the insect exoskeleton, tracheal system, and digestive
tract [[150]64,[151]65,[152]66,[153]67]. The inhibition of chitin
synthesis impedes the formation of the insect exoskeleton, disrupts the
molting processes, and can ultimately result in death
[[154]68,[155]69,[156]70]. However, the inhibition of chitin synthesis
is influenced by various factors, including the activities of several
enzymes—trehalose (Tre), glucose-6-phosphate isomerase (G6PI),
fructose-6-phosphate aminotransferase (GFAT), glucosamine-6-phosphate
N-acetyltransferase (GNA), phosphoglucosamine mutase (PAGM), and
UDP-N-acetylglucosamine pyrophosphorylase (UAP)—which are involved in
converting trehalose into chitin [[157]71,[158]72,[159]73]. For
example, Liu et al. (2015) [[160]74] found that cadmium stress can
stimulate the strong expression of the GFAT protein in the
hepatopancreas, thereby regulating chitin biosynthesis. Another
possible factor is the availability of raw materials for chitin
synthesis in the hemolymph, such as glycogen and trehalose, which can
lead to the blockage of chitin synthesis. The relative contents of
various substances of S. litura on ZnSO[4] and CK diets were
significantly higher than those on ZnCl[2], except for glutamine
([161]Figure 4). Glutamine mainly acts as an ammonia donor in the
synthesis of chitin and irreversibly converts fructose-6-phosphate into
glucosamine-6-phosphate under the catalysis of fructose-6-phosphate
amidotransferase [[162]75]. This indicates that glutamine is not
consumed but rather accumulates in the hemolymph during the chitin
synthesis process [[163]76]. Therefore, S. litura expends more energy
to resist the effects of Zn^2+ stress, leading to a reduction in
trehalose in the hemolymph, which, in turn, hinders the synthesis of
chitin and ultimately results in a prolonged developmental period or
the death of the insect.
The formation of free radicals constitutes the primary toxic effect of
metals on organisms, as these radicals can induce DNA damage, alter
sulfhydryl homeostasis, and promote lipid peroxidation [[164]77].
Numerous vitamins have been demonstrated to mitigate the physiological
damage resulting from an excessive intake of metal minerals
[[165]78,[166]79,[167]80]. For example, vitamin C and vitamin E, acting
as reducing agents, inactivate free radicals, thereby mitigating the
oxidative damage caused by heavy metals to organisms and providing
antioxidant protection against oxidative stress [[168]81]. Vitamin C
(ascorbic acid) has four hydroxyl groups that can bind to metal
substances, making vitamin C a metal oxide surface modifier [[169]82].
For example, Farjan et al. (2012) [[170]79] and Garg and Mahajan (1994)
[[171]83] have demonstrated that dietary vitamin C can increase
antioxidant activities such as catalase and glutathionease. Therefore,
fruits and vegetables rich in vitamin C possess antioxidant properties
and can reduce the damage caused by heavy metal poisoning [[172]80].
Similarly, vitamin E, a lipid-soluble non-enzymatic antioxidant,
inhibits reactive oxygen species (ROS) production, scavenges hydroxyl
radicals, and protects cells from lipid oxidation, mitigating
metal-induced damage in vitro and in animals with high concentrations
of iron, copper, and cadmium [[173]84,[174]85]. For example, Coskun et
al. (2020) [[175]78] demonstrated that dietary vitamin E reduces SOD
activity and MDA level in Galleria mellonella hemolymph, thereby
playing a protective role in selenium poisoning.
5. Conclusions
High concentrations of dietary ZnCl[2] (100 mg/kg, 200 mg/kg, and 300
mg/kg) significantly prolonged the preadult period and reduced the
preadult survival rate of S. litura, whereas a lower concentration of
dietary ZnCl[2] (50 mg/kg) significantly increased the fecundity of S.
litura. Additionally, dietary ZnSO[4] exerts a devastating effect on
the survival of S. litura. Even at the lowest concentration of 50 mg/kg
dietary ZnSO[4], only 1% of S. litura was able to complete the entire
life cycle. High-throughput untargeted metabolomics demonstrated that
both 100 mg/kg dietary ZnCl[2] and ZnSO[4] decrease the content of
hemolymph vitamins and increase vitamin C levels, thus helping S.
litura larvae counteract the stress induced by ZnCl[2] and ZnSO[4].
Simultaneously, dietary ZnCl[2] obstructs the chitin synthesis pathway
in the hemolymph of S. litura, thus extending the developmental period
of S. litura larvae. These results indicate that the effectiveness and
toxicity of zinc depend on its chemical form and concentration. Low
concentrations of Zn^2+ positively affect populations of S. litura, and
high concentrations of Zn^2+ negatively affect the population
performance and the hemolymph metabolism. Additionally, dietary
SO[4]^2- exerts a devastating effect on the S. litura population.
Acknowledgments