New Insights into the Life History Changes Can Enhance Control Strategies for Therioaphis trifolii

Abstract

The spotted alfalfa aphid (SAA), Therioaphis trifolii, is a phloem-feeding pest with a complex life history, and it causes a tremendous global loss of crop yields. A large number of previous studies focused only on few-generation life tables, whereas multi-generation life tables could explore aphid adaptation processes that are poorly understood. In this study, we investigated the effects of physical characters of alfalfa on SAA growth and development and used life-table parameters to evaluate the transgenerational effects of SAA populations on highly resistant (HA-3) and highly susceptible (Hu) alfalfa cultivars. The results indicated that alfalfa waxy content, anatomical structure of vegetative organs, and density and length of leaf hairs were significantly positively correlated with resistance to the SAA. In terms of the developmental time of total preoviposition period (TPOP), no significant differences were observed between two populations; compared to the HA-3 population, the G2-G8 Hu populations were markedly lower and G9-G10 Hu populations were visibly higher. Meanwhile, the reproductive days, mean longevity, and fecundity (offspring) of the HA-3 population were obviously higher than those of the Hu group after G5. Furthermore, the finite rate of increase (λ) and intrinsic rate of increase (r) of HA-3 were significantly higher than for the Hu population after G7. Meanwhile, the net reproductive rate (R₀) and mean generation time (T) of HA-3 were significantly higher than for the Hu population after G5. On the whole, the SAA had a high survival rate, strong reproductive capacity, long life span, and high population growth parameters on Hu in the early stage, while the SAA had better growth and development on HA-3 in the late stage. The physical characteristics of alfalfa leaves could be used as one of the indicators of aphid resistance. However, the coevolutionary coupling was broken with the gradual adaptation of SAA, which provides an empirical basis for further exploring the mechanisms of alfalfa resistance to aphids and the integrated control of pests.

1. Introduction

The spotted alfalfa aphid (SAA), Therioaphis trifolii Monell (Hemiptera: Aphididae), is a known destructive phloem-feeding economic pest with a complex life history, complex multi-productivity, and numerous endosymbionts [1]. The SAA was first described by Monell in 1882 in eastern North America [2], and it was first found on alfalfa in New Mexico, USA, in 1954 [3]. Following that, it began to appear in China as traders introduced high-quality species of lucerne from other continents [4]. It is one of the most damaging insect pests for alfalfa and has now spread worldwide by sucking the sap and tender stems and transmitting phytopathogenic viruses, which can easily cause plant nutrient deficiency, plant malformation growth, falling leaves, wilting, and even death [5,6]. The SAA severely inhibits seedling establishment and plant growth, and it affects forage quality and yield, especially for hay, with an estimated 10–30% loss of yield [6,7]. Determining how to effectively control the SAA has become one of the important issues in the development of the alfalfa industry. Although various management technologies including chemicals [8,9], farm management techniques, such as cutting and grazing [10], and biological control [11,12,13] have been developed and implemented to control the SAA, its management still primarily relies on the application of insecticides. Research has demonstrated that dependence on insecticides (demeton-S-methyl, dimethoate, pirimicarb, etc.) for SAA control could lead to the development of resistance, even if populations are generally much lower than they were in the late 1970s and early 1980s [8]. As better approaches, breeding and selecting aphid-resistant cultivars are the most economical and practical choices to control the alfalfa aphid, increase the forage yield, maintain quality, improve the stand persistence, and promote the economic development and ecologically benign development of western China [14].

Plant pest resistance is the best measure to affect and control pests through the morphological, organizational, and growth characteristics of the plant itself, such as the plant epidermal wax, leaf surface villi, leaf thickness, and cuticle, making these an important part of the pest management system [15,16,17]. Sa et al. compared the anatomical structures of vegetative organs of two different alfalfa cultivars, and the study showed that the tissue structure of ‘Caoyuan No. 4 alfalfa’ had obvious insect resistance characteristics, with its tissue resistance clearer than in ‘Caoyuan No. 2 alfalfa’ [18]. Over the years, researchers have used various methods to assess cultivar resistance, such as Wu et al., who used the aphid damage index to compare the resistance of different alfalfa cultivars to aphids [19]. Zhu et al., taking a different approach, used the population trend index method to study cucumber cultivars’ resistance to Aphis gossypii [20]. Pan et al., meanwhile, used the aphid resistance rate, susceptibility index, and improved aphid index evaluation as different methods to evaluate the resistance of 14 alfalfa cultivars to aphids [21]. Thackray et al., meanwhile, used the intrinsic rate of the increase of aphid populations on plants as an indicator to assess the resistance of wheat cultivars [22]. Hesler et al. then evaluated the resistance of wheat cultivars using the aphid number, development duration, and growth rate [23]. The development of resistance is an evolutionary response of insect pests to plant resistance. Compared to the SAA on susceptible plants, insects on resistant plants may have certain differences in their morphological characteristics, physiological responses, and biological characteristics. Accordingly, studies found significantly correlated differences between alfalfa cultivars and the growth and reproduction of the SAA [24,25]. Life-table analysis has emerged as a powerful tool for analyzing and understanding the effects of abiotic and biotic factors such as temperature on insect growth, survival, reproduction, and population growth [26,27,28]. This analysis is the key to population ecology [29], pest biological control [30], host preferences [25,31,32], and the adaptation of insects [28,33].

Much evidence suggests that Gannong No. 5 (HA-3) is a highly aphid-resistant cultivar bred by Gansu Agricultural University, and Hunter River (Hu) is a highly susceptible aphid cultivar introduced from Australia [25,34,35,36]. Slight changes in the morphological structure of alfalfa can change its palatability for the SAA, thereby affecting its life behavior, growth, and development. The present study aimed to evaluate transgenerational life-history changes associated with SAA populations on highly resistant and highly susceptible alfalfa cultivars through life-table analyses. We hypothesized that the cultivar with higher structural defense would be less suitable for SAA growth and reproduction, resulting in slower population growth of the SAA. However, SAAs might be expected to overcome resistance mechanisms and tend to improve their performance on resistant plants.

2. Materials and Methods

2.1. Insects and Plants

Two alfalfa cultivars were tested in this study, Gannong No.5 (HA-3) and Hunter River (Hu). HA-3 is a highly aphid-resistant cultivar bred by Gansu Agricultural University, and Hu is a highly susceptible aphid cultivar introduced from Australia. The SAA was originally collected from an alfalfa field at the research base of the School of Agriculture, Ningxia University. They were reared on fresh leaves from alfalfa cultivars HA-3 and Hu, which were placed in Petri dishes (9 cm diameter, 1.5 cm height). The insects were kept in a controlled insectarium at 25 ± 1 °C, 60 ± 5% relative humidity (RH), under a 16 h light/8 h dark photoperiod. Leaves were replaced every three days.

2.2. Determination of Epidermis Wax, Density, and Length of Leaf Hairs in Leaves of Two Alfalfa Cultivars

The leaves of two alfalfa cultivars with the same growth were randomly selected. After the leaves were washed and dried, the epidermis wax content was determined by the chloroform method [37], and the wax content was calculated by the unit fresh leaf weight. The leaf hair density and length were observed and measured using Leica DMC 4500 microscopy combined with Leica Application Suite 4.12.0 software (with six repetitions per variety).

2.3. Anatomical Structure of Vegetative Organs of Two Alfalfa Cultivars

The leaves with the same growth condition were randomly selected, and temporary slides were made by freehand sectioning. The anatomical structures of HA-3 and Hu stems and leaves were observed with an Olympus BX53 microscope, then photographed and measured with the software cellSens 1.12 (with six biological repetitions per variety).

2.4. Life Table

The adults were raised individually on HA-3 and Hu leaves. The petiole of each leaf was wrapped with absorbent cotton and placed abaxial side facing upward on moistened filter paper in a plastic Petri dish (9 cm diameter × 1.5 cm height). After 24 h, the adult aphid and other nymphs were removed, and only one nymph remained on the leaves of HA-3 and Hu; at least 60 neonate nymphs (i.e., 60 replicates) were observed in each population individually. Population parameters such as the development time of every stage, the spawning time, fecundity of each female, the number of reproductive days, longevity, and survival were recorded every 12 h. The number of nymphs molting and found dead were counted every 12 h until they became adults. After that, the number of nymphs produced per female in the aphid productive stage and the death time for each adult were counted every 12 h until all the adult aphids died. The newborn nymphs of each generation were removed as the next generation and raised in the same way for a total of 10 generations.

2.5. Statistical Analyzes

To determine the similarities or significant differences between the two populations, all parameters were analyzed using SPSS software (V.21.0, SPSS, Chicago, IL, USA) at a significance level of 0.05 using Student’s t-test. All raw life history data and population parameters for the SAA, HA-3, and Hu individuals were analyzed according to the age-stage, two-sex life-table theory, and methods [38,39] using the computer program TWOSEX-MSChart [40]. Population growth parameters included the development time of every stage, adult preoviposition period (APOP) (i.e., the duration from adult to the first oviposition), total preoviposition period (TPOP) (i.e., the duration from birth to the first oviposition), reproductive days, longevity, fecundity, age-stage specific survival rate (s_xj) (representing the probability that a newborn nymph would survive to age x and stage j where x is the age in days and j is the stage), age-stage specific fecundity (f_xj) (which represents the number of hatched eggs produced by a female adult at age x), age-specific survival rate (l_x), age-specific fecundity (m_x), intrinsic rate of increase (r), net reproductive rate (R₀), finite rate of increase (λ), mean generation time (T), and gross reproduction rate (GRR). To calculate the survival rate and fecundity of individuals at age x, the l_x and m_x were calculated as follows:

$$l_x={\displaystyle \sum }_{j=1}^k{s}_{xj}$$

$$m_x={\displaystyle \sum }_{j=1}^k{s}_{xj}{f}_{xj}/{\displaystyle \sum }_{j=1}^k{s}_{xj}$$

where k is the number of stages. GRR is the sum of the age-specific fecundity (m_x) of all populations, ignoring the survival rate.

$$GRR={\displaystyle \sum }_{x=0}^{\infty }{m}_x$$

The R₀ is defined as the total number of offspring that an individual can produce during its lifetime, which was calculated as follows:

$$R_0={\displaystyle \sum }_{x=0}^{\infty }{l}_x{m}_x$$

The r of increase was calculated using the Lotka–Euler equation with age indexed from zero, as:

$${\displaystyle \sum }_{x=0}^{\infty }{e}^{-r\left(x+1\right)}{l}_x{m}_x=1$$

The λ of increase was calculated as follows:

$$\lambda =e^r$$

The T is defined as the length of time a population requires to increase to R₀-fold of its size as time approaches infinity and the population settles down to a stable age-stage distribution. The mean generation time is calculated as follows:

$$T=\frac{ln{R}_0}{r}$$

The standard errors of all life-history traits and population parameters were calculated using the bootstrap method with 100,000 resamples [41,42]. Differences between treatments were compared by using the Tukey–Kramer procedure. All graphs were created using Origin 9.1 software.

3. Results

3.1. Determination of Epidermis Wax, Density, and Length of Leaf Hairs in Leaves of Two Alfalfa Cultivars

The epidermis wax contents of HA-3 and Hu leaves were 111.45 ± 12.99 and 24.20 ± 3.30 mg/g, respectively, with a significant difference between the two (p < 0.05). The density and length of leaf hairs in the leaves of the two alfalfa cultivars were also significantly different (Figure 1). Among them, the maximum leaf hair density and leaf length for HA-3 were 6.3/mm² and 1.22 mm, respectively. The study found that HA-3 had a mix of long and short leaf hairs, while Hu had only sparse longish leaf hairs.

3.2. Anatomical Structure of Vegetative Organs of Two Alfalfa Cultivars

The stems’ anatomical structures for HA-3 and Hu are presented in

Table 1. Comparison of stem anatomical structures between HA-3 and Hu.

Cultivars	Epidermal Thickness/μm	Thick Corner Tissue Thickness/μm	Thickness of Cortical Parenchyma Cells/μm	Phloem Thickness/μm	Cambium Thickness/μm	Xylem Thickness/μm	Pith Diameter Length/μm
HA-3	12.81 ± 0.29 b	26.22 ± 0.52 b	23.41 ± 0.55 b	16.44 ± 0.40 a	8.81 ± 0.35 a	76.76 ± 1.87 b	523.56 ± 1030 b
Hu	10.87 ± 0.25 a	21.36 ± 0.37 a	21.32 ± 0.47 a	14.76 ± 0.44 a	7.75 ± 0.27 a	60.75 ± 0.93 a	438.62 ± 6.92 a

Values in the table represent the mean ± SE. Different lower-case letters (a and b) indicate significant differences between the HA-3 and Hu groups at the level of p < 0.05, respectively (Student’s t-test).

. The results showed that the tissue structures of the HA-3 stem were larger than for Hu and significantly different, except for the phloem and cambium thicknesses. Similarly, when we compared the epidermis, mesophyll, and vein structures of HA-3 and Hu, the anatomical structures of HA-3 were larger than those of Hu, with significant differences in the palisade tissue, spongy tissue, cortical parenchyma cell, and xylem thicknesses (

Table 2. Comparison of epidermis and mesophyll structures between HA-3 and Hu.

Cultivars	Upper Epidermal Thickness/μm	Lower Epiderma Thickness/μm	Palisade Tissue Thickness/μm	Spongy Tissue Thickness/μm	Proportion of Palisade vs. Spongy Tissue
HA-3	9.710 ± 0.40 a	10.03 ± 0.25 a	32.40 ± 0.61 b	27.41 ± 0.89 b	1.23 ± 0.44 a
Hu	9.707 ± 0.22 a	9.39 ± 0.24 a	30.24 ± 0.46 a	25.07 ± 0.47 a	1.22 ± 0.32 a

Values in the table represent the mean ± SE. Different lower-case letters (a and b) indicate significant differences between the HA-3 and Hu groups at the level of p < 0.05, respectively (Student’s t-test).

and

Table 3. Comparison of leaf vein structure between HA-3 and Hu.

Cultivars	Leaf Vein Thick Corne Tissue Thickness/μm	Thickness of Cortical Parenchyma Cells/μm	Phloem Width/μm	Cambium Thickness/μm	Xylem Thickness/μm
HA-3	50.66 ± 1.00 a	15.76 ± 0.39 b	12.79 ± 0.92 b	5.96 ± 1.15 a	39.88 ± 2.16 b
Hu	49.77 ± 1.17 a	9.95 ± 0.65 a	9.49 ± 0.45 a	5.85 ± 0.28 a	27.26 ± 1.14 a

Values in the table represent the mean ± SE. Different lower-case letters (a and b) indicate significant differences between the HA-3 and Hu groups at the level of p < 0.05, respectively (Student’s t-test).

).

3.3. Developmental Time, Longevity, and Fecundity

The different immature instar development times, ATOPs, TPOPs, reproductive days, longevity, and fecundity for SAA G1-G10, for the HA-3 and Hu cultivars, are summarized in

Table 4. Developmental time and fecundity of SAA on HA-3 and Hu cultivars.

Generation	Cultivar	1st Instar (d)	2nd Instar (d)	3rd Instar (d)	4th Instar (d)	APOP (d)	TPOP (d)	Reproductive Days (d)	Mean Longevity (d)	Fecundity (Offspring)
G1	HA-3	1.62 ± 0.062 a	1.77 ± 0.071 a	1.53 ± 0.069 a	1.85 ± 0.098 a	0.46 ± 0.031 a	7.14 ± 0.163 a	3.74 ± 0.351 a	11.144 ± 0.365 a	13.88 ± 1.463 a
	Hu	1.70 ± 0.067 a	1.82 ± 0.067 a	2.14 ± 0.087 b	1.97 ± 0.121 a	0.40 ± 0.017 a	7.99 ± 0.173 a	3.88 ± 0.304 a	12.010 ± 0.378 a	17.37 ± 1.729 a
G2	HA-3	1.98 ± 0.077 b	2.03 ± 0.079 a	2.07 ± 0.090 a	2.04 ± 0.109 a	0.53 ± 0.039 a	8.43 ± 0.152 a	7.41 ± 0.904 a	16.816 ± 0.950 a	27.12 ± 4.009 a
	Hu	1.68 ± 0.041 a	1.78 ± 0.063 a	1.98 ± 0.084 a	2.02 ± 0.097 a	0.46 ± 0.024 a	7.51 ± 0.118 a	12.51 ± 1.094 b	19.719 ± 0.954 b	50.67 ± 4.885 b
G3	HA-3	1.48 ± 0.056 a	1.51 ± 0.058 a	1.82 ± 0.071 a	2.07 ± 0.101 a	0.51 ± 0.034 a	6.93 ± 0.124 a	10.82 ± 1.124 a	16.264 ± 0.874 a	41.76 ± 4.103 a
	Hu	1.69 ± 0.048 a	1.30 ± 0.055 a	1.60 ± 0.078 a	1.87 ± 0.081 a	0.55 ± 0.042 a	6.40 ± 0.091 a	14.19 ± 0.777 b	20.914 ± 0.788 b	70.86 ± 3.767 b
G4	HA-3	1.63 ± 0.039 a	1.71 ± 0.062 a	1.91 ± 0.075 a	2.13 ± 0.096 a	0.46 ± 0.024 a	7.56 ± 0.096 a	9.63 ± 0.802 a	16.384 ± 0.713 a	40.63 ± 3.401 a
	Hu	1.73 ± 0.063 a	1.79 ± 0.061 a	1.73 ± 0.072 a	1.94 ± 0.084 a	0.47 ± 0.034 a	6.81 ± 0.087 a	12.44 ± 0.667 b	19.191 ± 0.552 b	59.56 ± 2.740 b
G5	HA-3	1.83 ± 0.069 a	1.50 ± 0.064 a	1.83 ± 0.080 a	2.14 ± 0.092 a	0.46 ± 0.021 a	7.38 ± 0.103 a	11.36 ± 0.562 a	18.824 ± 0.648 a	45.70 ± 2.599 a
	Hu	1.86 ± 0.062 a	1.53 ± 0.061 a	1.92 ± 0.073 a	1.90 ± 0.079 a	0.41 ± 0.026 a	7.35 ± 0.077 a	13.02 ± 0.681 a	20.000 ± 0.664 a	51.90 ± 2.943 b
G6	HA-3	1.74 ± 0.058 a	1.49 ± 0.058 a	1.64 ± 0.057 a	1.86 ± 0.052 a	0.30 ± 0.014 a	6.73 ± 0.068 a	9.22 ± 0.693 a	15.700 ± 0.579 a	40.25 ± 2.280 a
	Hu	1.61 ± 0.041 a	1.39 ± 0.052 a	1.67 ± 0.059 a	1.99 ± 0.083 a	0.37 ± 0.024 b	6.70 ± 0.095 a	8.08 ± 0.506 a	14.873 ± 0.452 a	37.42 ± 1.981 a
G7	HA-3	1.62 ± 0.036 a	2.02 ± 0.089 b	1.87 ± 0.056 a	1.86 ± 0.060 a	0.35 ± 0.020 a	7.32 ± 0.101 a	11.39 ± 0.793 a	18.707 ± 0.755 a	47.53 ± 4.057 a
	Hu	1.58 ± 0.040 a	1.52 ± 0.088 a	1.73 ± 0.105 a	2.11 ± 0.093 b	0.43 ± 0.029 b	6.97 ± 0.135 a	11.29 ± 0.674 a	18.167 ± 0.741 a	47.23 ± 3.513 a
G8	HA-3	1.68 ± 0.046 a	1.40 ± 0.066 a	1.75 ± 0.065 a	1.88 ± 0.077 a	0.38 ± 0.024 a	6.93 ± 0.084 a	8.70 ± 0.933 b	15.730 ± 0.782 b	44.83 ± 4.808 b
	Hu	1.53 ± 0.052 a	1.42 ± 0.069 a	1.56 ± 0.080 a	1.87 ± 0.103 a	0.38 ± 0.021 a	6.84 ± 0.064 a	5.63 ± 0.450 a	12.570 ± 0.518 a	20.43 ± 1.833 a
G9	HA-3	1.83 ± 0.069 a	1.92 ± 0.076 a	2.23 ± 0.089 a	2.00 ± 0.102 a	0.67 ± 0.066 a	8.24 ± 0.108 a	3.31 ± 0.329 b	11.660 ± 0.247 a	18.89 ± 0.732 b
	Hu	1.86 ± 0.081 a	2.00 ± 0.103 a	2.16 ± 0.142 a	2.07 ± 0.151 a	0.69 ± 0.051 a	8.75 ± 0.170 a	2.56 ± 0.162 a	11.037 ± 0.452 a	10.06 ± 1.251 a
G10	HA-3	2.13 ± 0.080 a	1.90 ± 0.125 a	1.72 ± 0.152 a	2.10 ± 0.192 a	0.72 ± 0.125 a	9.06 ± 0.242 a	3.20 ± 0.327 b	12.792 ± 0.571 a	14.13 ± 1.693 b
	Hu	2.33 ± 0.112 a	1.81 ± 0.124 a	1.76 ± 0.166 a	1.69 ± 0.208 a	0.69 ± 0.114 a	9.25 ± 0.231 a	2.00 ± 0.332 a	11.278 ± 0.529 a	5.71 ± 1.165 a

Values in the table represent the mean ± SE. Different lower-case letters (a and b) indicate significant differences between the HA-3 and Hu groups at the level of p < 0.05 in the same generation, respectively (Student’s t-test). APOP, adult pre-reproductive period (d); TPOP, total pre-reproductive period (d).

. The immature instar developmental times, ATOPs, and TPOPs varied between the strains (except for a few periods), but there was no significant difference (p > 0.05); compared to the HA-3 population, the G2-G8 Hu populations were markedly lower and the G9-G10 Hu populations were visibly higher. For the reproductive days, mean longevity, and fecundity (offspring), the G1-G5 Hu populations were obviously higher, while the G6-G10 Hu populations were obviously lower than the HA-3 group. Meanwhile, the results showed an increase in G1-G5 and reduction in G6-G10 between the two populations as a whole.

3.4. Population Growth Parameters

The population growth parameters were estimated using bootstrapping methods based on the life table. The GRR, λ, r, R₀, and T for G1-G10 SAAs were calculated and analyzed, as presented in

Table 5. Population parameters of SAA on HA-3 and Hu cultivars.

Generation	Cultivar	GRR (Offspring/Individual)	λ (d−1)	r (d−1)	R0 (Offspring/Individual)	T (d)
G1	HA-3	29.589 ± 0.009 a	1.275 ± 0.0001 b	0.243 ± 0.0001 b	10.217 ± 0.0051 b	9.565 ± 0.0012 a
	Hu	31.014 ± 0.018 b	1.201 ± 0.0001 a	0.183 ± 0.0001 a	7.017 ± 0.0052 a	10.642 ± 0.0012 b
G2	HA-3	81.663 ± 0.034 a	1.275 ± 0.0003 a	0.243 ± 0.0001 a	24.450 ± 0.0052 a	13.147 ± 0.0013 b
	Hu	110.965 ± 0.034 b	1.366 ± 0.0003 b	0.312 ± 0.0001 b	46.300 ± 0.0052 b	12.300 ± 0.0012 a
G3	HA-3	109.606 ± 0.033 b	1.386 ± 0.0001 a	0.326 ± 0.0001 a	35.183 ± 0.0046 a	10.917 ± 0.0009 a
	Hu	105.154 ± 0.031 a	1.471 ± 0.0002 b	0.386 ± 0.0001 b	69.750 ± 0.0045 b	11.002 ± 0.009 b
G4	HA-3	82.979 ± 0.026 b	1.363 ± 0.0002 a	0.310 ± 0.0002 a	33.117 ± 0.0062 a	11.299 ± 0.0013 b
	Hu	80.685 ± 0.025 a	1.451 ± 0.0002 b	0.372 ± 0.0002 b	52.933 ± 0.0065 b	10.670 ± 0.0013 a
G5	HA-3	81.438 ± 0.034 b	1.381 ± 0.0001 a	0.323 ± 0.0001 a	40.433 ± 0.0054 a	11.466 ± 0.0011 a
	Hu	80.887 ± 0.033 a	1.384 ± 0.0001 b	0.325 ± 0.0001 b	44.983 ± 0.0055 b	11.722 ± 0.0012 b
G6	HA-3	80.523 ± 0.029 b	1.446 ± 0.0003 a	0.369 ± 0.0002 a	39.750 ± 0.0052 b	9.966 ± 0.0011 b
	Hu	73.513 ± 0.029 a	1.447 ± 0.0003 b	0.370 ± 0.0002 b	36.783 ± 0.0052 a	9.782 ± 0.0011 a
G7	HA-3	97.591 ± 0.022 b	1.364 ± 0.0002 a	0.312 ± 0.0001 a	42.917 ± 0.0044 b	12.100 ± 0.0012 b
	Hu	29.899 ± 0.019 a	1.370 ± 0.0002 b	0.314 ± 0.0001 b	35.733 ± 0.0044 a	11.366 ± 0.0012 a
G8	HA-3	75.591 ± 0.023 b	1.358 ± 0.0001 b	0.306 ± 0.0001 b	28.550 ± 0.0064 b	10.944 ± 0.0009 b
	Hu	49.899 ± 0.021 a	1.310 ± 0.0001 a	0.270 ± 0.0001 a	14.350 ± 0.0063 a	9.858 ± 0.0009 a
G9	HA-3	30.051 ± 0.008 b	1.217 ± 0.0001 b	0.196 ± 0.0002 b	8.033 ± 0.0044 b	11.498 ± 0.0012 b
	Hu	19.468 ± 0.005 a	1.179 ± 0.00001 a	0.161 ± 0.0002 a	7.050 ± 0.0041 a	9.944 ± 0.0011 a
G10	HA-3	23.064 ± 0.007 b	1.126 ± 0.0001 b	0.129 ± 0.0001 b	3.900 ± 0.0006 b	11.462 ± 0.0012 b
	Hu	22.546 ± 0.007 a	1.088 ± 0.0001 a	0.084 ± 0.0001 a	2.550 ± 0.0007 a	11.098 ± 0.0012 a

Values in the table represent the mean ± SE. Means followed by different letters significantly differ between the HA-3 and Hu groups in the same generation (p < 0.05, Student’s t-test). The standard errors of the parameters were analyzed by using the bootstrap technique in the TWOSEX-MSChart with 100,000 resamples. GRR, gross reproduction rate (offspring/individual); λ, finite rate of increase (day⁻¹); r, intrinsic rate of increase (day⁻¹); R₀, net reproductive rate (offspring/individual); T, mean generation time (day).

. The results showed that the life-table parameters were significantly different between the two populations. The GRR was significantly higher in the HA-3 group after G2. The λ and r were significantly increased in the G2-G7 Hu populations, and vice versa in the other generations. The R₀ was obviously higher in the G2-G5 Hu groups, and the opposite was seen in the other generations. The T showed no regularity from G1-G5, after which the HA-3 populations demonstrated significantly longer times than the Hu populations.

The age-specific survival rate (l_x) represented the probability of a newborn nymph surviving to age x, and l_x provided a simplified summary of the survival history of the whole life period (Figure 2). The l_x curves of the G1-G4 HA-3 populations showed a greater decline than for the Hu populations, but the opposite in the other generations. The age-specific fecundity (m_x) demonstrated the number of offspring produced by individuals at age x (Figure 3). The m_x peaks for the G1, and G2-G5 HA-3 populations were lower than for the Hu populations, and the inverse was true in the other generations. Based on the m_x curve, the highest age-specific fecundity peak for the Hu population (3.53 offspring/12 h) occurred at the age of G4 8.5 days. In contrast, the HA-3 population had a maximum peak at the age of G6 8.5 days (3.25 offspring/12 h). The age-specific reproductive value (V_x) indicated the reproduction dedication of an x-age individual to future generations (Figure 4). The change trend of V_x was similar to that of m_x. The maximum V_x of the G1, G3, and G4 Hu populations (14.13 (16 days), 16.21 (8 days), and 16.78 (8 days), respectively) were distinctly higher than those for the HA-3 populations (4.34 (9.5 days), 7.06 (8 days), and 7.18 (8 days), respectively). With the adaptation of the insects, the maximum V_x in the G8-G10 HA-3 populations was higher than that in the Hu populations. The age-specific life expectancy (e_x) displayed the expected life span of an individual of age x (Figure 5). The e_x curve exhibited how G1 and G2 Hu populations had a longer life expectancy than the HA-3 populations. The life expectancy was also longer in G3-G5 Hu populations than in HA-3 populations before 15 days. Yet, the life expectancy of HA-3 populations was longer than that of Hu populations after G7.

4. Discussion

In the long-term co-evolution of plants and insects, plants take a series of measures to resist the harm of insects through changes to their tissue structure, including wax, epidermal hairs, glands, cell wall thickness, lignin content on the plant surface, etc. [15,16,17]. Previous studies reported that leaf waxiness distinctly positively linearly correlated with resistance to Apolygus lucorum [43] and the SAA [44]. Similarly, our results showed that the alfalfa waxy content was significantly positively correlated with resistance to the SAA. In addition, our results showed that the upper and lower epidermal, palisade tissue, spongy tissue, and other tissue thicknesses of HA-3 were higher than for Hu. Similar results were found by Xiao et al., who demonstrated that the leaf thickness of cassava was distinctly negatively correlated with the mites damage index [45]. These findings indicated a close relationship with the insect resistance of HA-3, where certain physical traits have significant implications for insect resistance and thus could be targeted as traits for breeding.

The life table was a useful method for studying the survival, stage differentiation, and population dynamics of insects simultaneously; it was also crucial for furthering our knowledge about the population ecology and pest control. Breeding and selecting aphid-resistant cultivars were the most economical and safe ways to control alfalfa aphids and increase the alfalfa yield [14]. Lloyd et al. found that HA-3 was a highly aphid-resistant variety, while Hu was a highly susceptible variety introduced from Australia [34,35,36]. So, this study set out to determine different alfalfa cultivars’ effects on transgenerational life-history changes (for 10 successive generations, G1–G10) of the SAA. The trends in the life tables and population parameters confirmed that SAA populations were influenced by the alfalfa cultivars, which demonstrates the selective and adaptive process of SAA development, as studied on two quite different alfalfa cultivars.

Adaptation was a biological response to various environmental conditions [46]. Generally speaking, the development of resistance by a pest in the face of a resistant plant reduces its adaptation, and resistant populations were less competitive and had lower survival rates, longer developmental times, and lower fertility. In fact, the opposite was expected, since the development of resistance by a pest involves overcoming the resistance mechanisms of the plant and gradually regaining survival and reproduction.

According to the literature, most experiments to determine the plant resistance through insect life tables and population growth parameters were cultured for less than five generations [25,34,47,48]. In the present study, when we investigated the reproductive days, mean longevity, fecundity (offspring), and R₀ values, these were clearly higher for the G1-G5 Hu populations than for the HA-3 groups, which is consistent with previous reports [34]. Our results were also in line with a previous finding that the r_m values for the SAAs on susceptible plants were significantly higher than on resistant cultivars [49]. Further to this, our study showed that population parameters (l_x, m_x, V_x, and e_x) were higher in the G1-G4 Hu groups than in the HA-3 group as a whole (Figure 2, Figure 3, Figure 4 and Figure 5). This could be due to the differences in physical traits between the two alfalfa cultivars, for example, the waxy content and leaf thickness of HA-3 are higher and thicker than those of Hu, respectively. The epidermal hair acts as a natural physical barrier, which can effectively prevent pests, herbivores, and parasitic plants. The leaf hair length and density, and growth of aphids have been well-documented in studies that positively correlated the leaf hair length with the developmental duration of aphids, and those that negatively correlated the leaf hair density with the mean relative growth rate of aphids [50]. These results could also reflect how at every stage of plant growth and development, volatile substances and secondary metabolites such as aldehydes, aromatics, phenolic compounds, flavonoids, steroids, terpenoids, and alkaloids are released to deter insects from feeding [51,52,53]. Plants can also produce chemical signals, such as phytohormone jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), salicylic acid (SA), and other specific chemicals to interfere with pests’ physiology [54,55,56].

In addition to this, plant cells exhibited high responsiveness to various stresses through a flexible and finely balanced response network, including reduction–oxidation, stress hormones, Ca⁺ influxes, protein kinases (MAPKs), reactive oxygen species (ROS), and phytohormone (JA, ET, SA, etc.) signaling pathways [56,57,58,59]. However, different results were also reported previously, showing that the Hu cultivar may effectively resist damage from SAAs, as shown when SAAs were allowed to feed on 10 alfalfa cultivars [25]. This may be because insect life histories are impacted by changes to the host plant quality, with poor quality often associated with reduced herbivore fecundity, size, and longevity. The aphid’s developmental time, fecundity, individual size, and longevity were all related to the quality of the host plant [60]. Another reason might be that there were differences between the alfalfa cultivars selected in the study. If the cultivars in the comparison test were all aphid-susceptible, then Hu was the most aphid-resistant among them.

Some of the chemicals might change mate location, courtship, and oviposition of insects [61]. Interestingly, we found that alfalfa resistant to aphids had a positive impact on the oviposition and embryogenesis process of SAAs. In the present study, our study showed that the HA-3 population had significantly increased TPOPs, reproductive days, mean longevity, fecundity, R₀, and T in G6-G10. Simultaneously, the GRR was significantly higher in the HA-3 group after G2. Further, the results of our research indicated that λ and r significantly differed between the two populations, with the HA-3 population having significantly increased rates after G7. Similar results were found for the population parameters l_x, m_x, V_x, and e_x. The SAA could manipulate their host plants by using their styles to pierce the plant tissue, inject salivary secretions into the phloem, and expand rapidly under optimal conditions [62,63,64]. The secreted proteins might inhibit plant defense responses and thereby enable the SAA to effectively counteract the physiological and biochemical barriers of host plants [65,66]. For example, salivary oxidation might be a way for the alfalfa aphid to dispose of harmful phenolics in the sensitive host alfalfa [67]. In addition, insects could use the detoxification enzyme system in their gut and fat, such as cytochrome P450s (CYPs), to weaken anti-insect substance activity [68]. Certain studies have confirmed that aphids could form a symbiotic relationship with a variety of microorganisms, which support the host to perform a variety of biological functions, including improved resistance to biotic or abiotic stresses [69,70]. Our results might reflect how the Hu populations met with no initial resistance from the alfalfa, and then their population parameters dropped as the plants began using defense mechanisms. For the HA-3 populations, the initially strong defense mechanisms they met from the HA-3 cultivar came to be overcome from further adaptation of the SAA [25]. Therefore, the key factors for the increased fitness in the HA-3 populations need further study. At the same time, our results suggest that aphid control should be carried out in the early stage of aphid epidemic.

5. Conclusions

In conclusion, the results showed that the SAA had a high survival rate, strong reproductive capacity, long life span, and high population growth parameters on susceptible alfalfa in the early stage, while the SAA had better growth and development on resistant alfalfa in the late stage, which made the control of the SAA more challenging. At the same time, the results indicated that alfalfa’s initial physical resistance to aphids could not ensure its continued maintenance of resistance, a factor that should be considered from physiological, biochemical, and molecular perspectives. In summary, these results laid the foundation for further research on the growth and development of the SAA on alfalfa. Future research should further explore the defense mechanism of alfalfa and the anti-defense mechanism of the SAA. In recent years, with the extensive use of insecticides, insects have become resistant to many of these, and there are also hidden safety problems. In that context, studying the interactions between alfalfa and the SAA were useful to provide a scientific basis for the effective green prevention and control of the SAA, with the goal being its sustainable management.