The host range of Aphis gossypii is dependent on aphid genetic background and feeding experience

Introduction

Most insect herbivores have specialized diets (Jaenike, 1990; Via, 1991; Caballero, Ramirez & Niemeyer, 2001; Stone et al., 2009). Even polyphagous species show some degree of dietary specialization, and they often perform better on some host plants than on others. Particular insect herbivore populations may perform better on their natal hosts than on non-natal or novel hosts (Jaenike, 1990). The formation of host biotypes and the potential flexibility of dietary specialization within polyphagous species is not well understood. For example, host specialization in the polyphagous aphids, Acyrthosiphon pisum and Rhopalosiphum maidis, could not be modified by feeding experience on a novel host (Via, 1991; Caballero, Ramirez & Niemeyer, 2001). However, host specialization in other insect herbivore species was alterable (Singer, Thomas & Parmesan, 1993; Messina, Mendenhall & Jones, 2009; Wu et al., 2013). The introduction of a novel plant into a region led to a change in host availability for Euphydryas editha butterflies, and even resulted in a local population’s refusal to accept their ancestral hosts (Singer, Thomas & Parmesan, 1993). The seed beetles, Callosobruchus maculatus (F.), performed worse when they were fed on lentil than on mung bean, but after ten generations on lentil, their survival rate increased up to 88% which was similar to the value (97%) on mung bean, and consequently a self-sustaining population on lentil resulted (Messina, Mendenhall & Jones, 2009). These results illustrate that the diet of insect herbivores can change in response to resource availability (Cates, 1980).

The cotton-melon aphid, Aphis gossypii Glover, has a wide host range, perhaps 900 species belonging to 116 plant families, such as Cucurbitaceae, Malvaceae, Solanaceae, Rutaceae, and Asteraceae (Ebert & Cartwright, 1997; Blackman & Eastop, 2000). However, many studies illustrate that A. gossypii populations have formed obvious host races, which use only a subset of host plant species in their recorded host range (Kring, 1959; Guldemond, Tigges & De Vrijer, 1994; Liu, Zhai & Zhang, 2002; Liu, Zhai & Zhang, 2008; Liu et al., 2004). Numerous cases of host race, biotype, and host-specialized strains in A. gossypii populations have been found all over the world. In Europe, the aphid A. gossypii from chrysanthemum could not colonize cucumber and vice-versa, cucumber aphids did not use chrysanthemum (Guldemond, Tigges & De Vrijer, 1994). Five host races (cucurbits, cotton, eggplant, potato, and chili- or sweet-pepper races) were identified in A. gossypii populations collected from five plant families in five large geographical regions (Carletto et al., 2009). In China, A. gossypii populations on cotton could not colonize cucumber seedlings, and the populations collected from cucumber could not colonize cotton seedlings either (Liu, Zhai & Zhang, 2008; Wang et al., 2016). The populations on cucumber could not use chrysanthemum, but these on cotton could use this plant (Liu, Xu & Lei, 2017). These results indicate that A. gossypii populations have differentiated into at least two host biotypes: cotton- and cucurbit-specialized aphids.

Moreover, recent experimental data show that host specialization in A. gossypii populations is conditional. The host range expansion of host-specialized A. gossypii has been found under some specific conditions (Liu, Zhai & Zhang, 2008; Wu et al., 2013; Xu, Ma & Liu, 2014; Liu, Xu & Lei, 2017; Hu et al., 2017). Feeding experience on cowpea for one generation or on hibiscus plants for three generations extended the host range of the cucurbit-specialized biotype, and this biotype gained the ability to use both the cotton and cucumber seedlings (Liu, Zhai & Zhang, 2008; Wu et al., 2013). When the cotton-specialized biotype was reared for five or more generations on zucchini plants, it acquired the ability to use cucumber seedlings, but consequently lost the ability to use cotton plants (Wu et al., 2013). The cucumber at the inflorescence stage and cotton at the infructescence stage acted as a refuge for the cotton- and cucumber-specialized aphid biotypes, respectively, when these aphids encountered a food deficiency (Liu, Xu & Lei, 2017). These previous studies illustrate that the host range of aphid populations varies with the population’s feeding experience, and the host specialization of aphids to a specific host plant can change over a small number of generations.

Feeding experience, or host shifts, give aphids an opportunity to ingest some novel substances from new plants. Nutritional quality and secondary metabolites in plants affect the food quality of host plants for insect herbivores (Felton et al., 1989; Felton, 1996; Couture et al., 2016). Only the populations or species of insects adapted to the nutrients and the secondary metabolites of plants can survive and colonize the plant. That feeding experience on specific host plants shifted the host specialization of A. gossypii suggests that substances in host plants might determine the host range of aphids. How feeding experience over a small number of generations can maintain or change the host range of aphids is not clear.

On the other hand, genetic backgrounds of aphids may affect their host range. It has been found that the genetic structure of insect populations changed in association with the use of novel host plants (Singer, Thomas & Parmesan, 1993). High genetic differentiation between host races was found in the pea aphid (Jaquiéry et al., 2012). The main genotypes of A. gossypii on cotton and cucurbit plants were also different (Carletto et al., 2009; Wang et al., 2016; Liu, Xu & Lei, 2017).

In this study, we propose the hypothesis that the host range of the cotton-melon aphid is determined by aphid genetic background and feeding experience. Due to the coexistence of nutrition and defensive contents in plants, it is difficult to distinguish the roles of nutrition and secondary metabolites in aphid feeding experience. Therefore, experiments using artificial diets can reveal how feeding experience alters insect host range, because the contents in artificial diet are wholly controllable. In this study, we firstly confirmed that three aphid genotypes in A. gossypii populations collected from cotton did not establish populations on intact plants or excised leaves of cucumber, and the other three genotypes collected from cucumber did not establish populations on intact plants or excised leaves of cotton. Secondly we examined the performance of these six aphid genotypes on artificial diet without plant secondary metabolites, and found that all of them could complete their life cycles. And then, these six aphid genotypes reared on artificial diet were transferred onto cotton and cucumber excised leaves to examine their ability to use leaves of both plant species. Finally, the genotypes that established populations on both the cotton and cucumber leaves via feeding experience on artificial diet were transferred back onto the cotton and cucumber seedlings to examine the change in host range. The study addresses the role of feeding experience on artificial diet in determining aphid host range, and provides a better understanding of the formation of aphid host specialization.

Materials and Methods

Performance of aphids on artificial diet and excised leaves

The liquid artificial diet of aphids was prepared according to the method by Mittler & Dadd (1962) and Auclair (1967a), Auclair (1967b). The main ingredients of this artificial diet are amino acids, sugar and vitamin B, excluding any secondary metabolites in host plants (Table 2). Life tables of each aphid genotype on the artificial diet were established. During the experiment, 150 µl artificial diet was placed between two layers of thin Parafilm which was fixed to one end of a glass tube (25 mm in diameter and 30 mm height). Fifteen to twenty apterous adult aphids were transferred onto the Parafilm in the tube, and then the other end of the tube was covered with one layer of Parafilm to prevent escape of aphids (Fig. 1). The next day, all the adults in the tube were removed, and ten newly-born nymphs were maintained as the original cohort for establishing the population documented in the life table. The survival and reproduction of the cohort of aphids were surveyed every two days until they died. During the reproductive period, nymphs were counted and then removed. The artificial diet was replaced every three days. The life tables of six aphid genotypes were performed on the artificial diet with 4 or 5 replications.

Table 2:

Ingredient of the artificial diet in 100 ml H₂O.

Ingredient	Dosage/mg	Ingredient	Dosage/mg
Cholesterol	5.0	Sucrose	30,000.0
L-Arginine	400.0	KH2PO4	500.0
L-Threonine	200.0	MgCl2⋅6H2O	200.0
L-Leucine	200.0	Biotin (VB7)	0.2
L-Tryptophane	120.0	Folic acid	0.5
L-Aspartic acid	600.0	Riboflavin (VB2)	2.5
Glycine	100.0	Pyridoxol (VB6)	2.5
L-Tyrosine	20.0	Calcium pantothenate (VB5)	5.0
γ-aminobutyric acid	20.0	Choline chloride	50.0
L-Histidine	200.0	Thiamine hydrochloride (VB1)	2.5
L-Lysine	200.0	Nicotinic acid (VB3)	10.0
L-Phenylalanine	120.0	Inose	50.0
L-Asparagine	110.0	P-Aminobenzoic acid	10.0
L-Glutamin	600.0	Ascorbic acid	100.0
L-Serine	100.0	Ca(H2PO4)2	1.4
L-Cysteine	50.0	FeC6H5O7⋅5H2O	0.4
L-Methionine	120.0	MgSO4	1.4
L-Valine	200.0	CuCl2⋅2H2O	0.6
L-Isoleucine	150.0	ZnCl2	2.6
L-Glutamic acid	200.0	Ca(C3H5O3)2⋅5H2O	3.4
L-Alanine	100.0	NaH2PO4⋅2H2O	1.0
L-Proline	100.0	NaCl	0.6
L-Cystine	10.0	MnCl2⋅4H2O	1.0

DOI: 10.7717/peerj.7774/table-2

The life tables of three genotypes from cotton and three genotypes from cucumber were also performed on the excised cotton and cucumber leaves, respectively. The leaf petiole was wrapped in wet cotton wool to keep leaf fresh, and then the leaf was placed into a Petri dish. Five apterous adult aphids were transferred onto the leaf for producing offspring at night, and then the mother aphids were removed and ten new-born aphids were kept for surveying their survival and reproduction. The survey was performed every two days following the method used in the artificial diet experiment above. Five replications were performed for each genotype on the cotton and cucumber excised leaves. The intrinsic rate of natural increase (r_m) was used to indicate the performance of aphids on artificial diet or host leaves which was calculated by the formula: r_m = ln(R ₀)/T, where R ₀= Σ l_x m_x, T = Σxl_xm_x∕R₀, l_x is the proportion of individuals in the initial cohort alive at age x days, and m_x is the mean number of progeny produced per mother aphid alive on day x.

Results

Population establishment of different aphid genotypes on intact plants of cotton and cucumber

The aphids from intact cotton and cucumber plants did not establish populations on the alternative host plant. The three aphid genotypes collected from cotton CO-1, CO-2, and CO-3 established populations on cotton plants, but the three genotypes from cucumber CU-1, CU-2, and CU-3 did not establish populations on cotton (Fig. 2A). The aphid genotypes from cucumber survived 3-11 days on cotton plants (Fig. 2A). Similarly, the three aphid genotypes collected from cucumber CU-1, CU-2, and CU-3 established populations on cucumber plants, but genotypes from cotton CO-1, CO-2, and CO-3 did not establish populations on cucumber plants, and they survived only 3–7 days (Fig. 2B).

Population growth of different aphid genotypes on excised leaves of the alternative host plant

The aphids on cotton and cucumber did not establish populations on the excised leaves from the alternative host plant. The aphid populations of six genotypes increased well on the excised leaves from their natal host plants (Figs. 3A 3B), but the genotypes from cucumber survived 4–10 days on the excised cotton leaves (Fig. 3A), and the genotypes form cotton survived 25–37 days on the excised cucumber leaves (Fig. 3B).

Performance of different aphid genotypes on artificial diet

The artificial diet was not very suitable for the six aphid genotypes, compared to the host leaf. The intrinsic rates of natural increase (r_m) of aphid populations were significantly affected by food type (F_1,59 = 2, 316.782, P < 0.001) and aphid genotype (F_5,59 = 14.095, P < 0.001) (Fig. 4). The r_m of all six aphid genotypes on excised leaves from the natal host plant were significantly higher than that on the artificial diet (CO-1: t = 14.141, P < 0.001; CO-2: t = 14.265, P < 0.001; CO-3: t = 16.483, P < 0.001; CU-1: t = 29.805, P < 0.001; CU-2: t = 42.605, P < 0.001; CU-3: t = 17.075, P < 0.001) (Fig. 4). The r_m values on natal host leaves were 1.5 to 5.2 times higher than the values on the artificial diet for all six aphid genotypes. The r_m of CO-1 was significantly higher on the artificial diet than that of the other five genotypes, and the r_m of CU-3 was significantly higher than that of CO-2, CU-1, and CU-2 on artificial diet (F_5,24 = 42.634, P < 0.001) (Fig. 4).

Feeding experience on artificial diet affects host range of aphids

Feeding experience on artificial diet altered host plant range of some aphid genotypes. Three aphid genotypes from cotton CO-1, CO-2, and CO-3 still maintained the ability to use their natal host cotton leaf when they were conditioned to the artificial diet for seven days (Fig. 5A). Moreover, two aphid genotypes CO-2 and CO-3 acquired the ability to use cucumber leaf, an alternative host plant (Fig. 5A). The population sizes of CO-2 and CO-3 on excised cucumber leaves were significantly higher than that of CO-1, CO-2 and CO-3 on excised cotton leaves (F_5,54 = 181.42, P < 0.001; Fig. 5A). Although genotype CO-1 conditioned to artificial diet could survive a long time on excised cucumber leaves (34 days), it did not establish a population (Fig. 5A).

Three aphid genotypes from cucumber CU-1, CU-2, and CU-3 established populations on excised cucumber leaves after they fed on artificial diet for seven days (Fig. 5B). Interestingly, one aphid genotype (CU-3) established a population on excised cotton leaves too after feeding experience on artificial diet, and the population size of this genotype on cotton leaves was significantly larger than those of genotypes CU-1 and CU-2 ( F_5,49 = 126.91, P < 0.001) (Fig. 5B). Genotypes CU-1 and CU-2 could survive more than 100 days on cotton leaves after feeding experience on artificial diet, but eventually all of them died (Fig. 5B).

Feeding experience on the excised leaf affects aphid host range

Host range expansion of aphids mediated by feeding experience was genotype-dependent. The aphid genotypes CO-2 and CO-3 established populations on the excised leaves of cucumber after feeding experience on artificial diet. However, after they were reared for 12 successive generations on the cucumber leaves, both genotypes still could use cotton plants, but could not use cucumber plants (Fig. 6A). By contrast, the genotype CU-3 fed on excised cotton leaves for 12 generations after experience on artificial diet could use both the cucumber and cotton plants (Fig. 6B), suggesting the expansion of host range. The population growth of these CU-3 on cotton plants was faster in one replicate after 25 days than in the other two, and therefore, the average number of aphids showed a high variance (Fig. 6B).

Discussion

Phytophagous insects, including dietary generalist species, often exhibit host specialization (Futuyma & Moreno, 1988; Jaenike, 1990; Sword, Joern & Senior, 2005; Stilmant et al., 2008). However, several factors, including both evolutionary and ecological processes, may induce changes in the host range of insect herbivores, and result in the acquisition or loss of specific host plants in their host range (West-Eberhard, 1989; Gorur, Lomonaco & Mackenzie, 2007). The host specialization of phytophagous insects is not wholly conservative (Nosil, 2002; Gorur, Lomonaco & Mackenzie, 2005; Liu, Zhai & Zhang, 2008). The results of the present study indicate that some aphid genotypes acquired the ability to survive and establish populations on excised leaves of a novel host plant which they did not use, after they underwent a feeding experience on artificial diet, but some genotypes did not acquire this ability. A previous study also found that a specific host plant could lead to a host shift of the host-specialized aphids (Wu et al., 2013). Because the experimental plants included both nutrients and secondary metabolites, it was unclear how each of these substances or if both contributed to the host shift of aphids. In this study, we found that the artificial diet lacking in secondary metabolites could alter the host range of a part of genotypes in A. gossypii aphids from cotton and cucumber. This result shows that feeding experience with nutrients alone can contribute to a change in host range of some aphid genotypes. Aphids feeding on different host plants may ingest different nutrients, and each genotype may adapt specifically to this nutrient profile (Via, 1991; Carletto et al., 2009; Alkhedir et al., 2016). Further study is needed to determine which specific nutrients are involved.

Plant resistance to aphids is related to host quality in terms of free amino acids composition, phytohormone content (abscisic acid, jasmonic acid, salicylic acid), and secondary metabolite profiles (Kant et al., 2004; Schilmiller & Howe, 2005; Chiozza, O’Neal & MacIntosh, 2010; Vos et al., 2013; Agut et al., 2014; Agut et al., 2016). Nutritional quality and secondary metabolite profiles vary dramatically between the natal and alternative host plants for aphids (Wang et al., 2015; Wang et al., 2017). As key nutrients for insects, free amino acids in plants are closely related to insect feeding, development, and reproduction (Mittler, 1970; Wilkinson & Douglas, 2003). Within a host plant species, phenotypic variation, sometimes characterized as plant vigor, affects the performance of aphids (Price, 1991; White, 2009); variation in plant vigor would result from variation in nutrients and secondary metabolites. In previous studies, we found that the cucurbit-specialized A. gossypii aphids could not survive on vigorous cotton plants, but they could colonize the old cotton plants by feeding on their old leaves. In contrast, the cotton-specialized aphids preferentially chose the vigorous seedlings of cotton and fed on the new leaves (Xu, Ma & Liu, 2014; Liu, Xu & Lei, 2017). In this study, the results indicate that the excised leaves and intact plants have different effects on performance of aphids. Other studies point to some of the specific differences between excised leaves and intact plants. The excised leaves of corn seedlings produced more jasmonic acid- and volicitin-induced sesquiterpene volatile than intact plants (Schmelz, Alborn & Tumlinson, 2001). Leaf excision stimulated increased ethylene production in rice leaves (Tsai, Hung & Kao, 1996). The amino acid content in the excised turnip leaves was higher than that in the intact leaves (Thomas, Sorensen & Painter, 1966). Plant secondary metabolites and nutrients have a strong impact on consumption and growth in dietary generalist herbivores, such as the cabbage looper Trichoplusia ni (Kaplan, McArt & Thaler, 2014). Excised leaves and old plants might have reduced defensive traits and improved nutrients to aphids compared to vigorously growing tissues, so aphids from other hosts might survive on them. For example, survival of spotted aphids was higher on excised than intact leaves of alfalfa (Thompson, Stewart & Morris, 1966). However, in this study, we also found that an artificial diet lacking secondary metabolites not only changed performance of aphids, but also induced host range expansion of these aphids. The result indicates that just host nutrients and not the secondary metabolites which aphids ingest from food can affect aphid host range. We speculated that the cucurbit-specialized and cotton-specialized biotypes in A. gossypii populations might result from different nutrient profiles, such as amino acids, in cotton and cucumber plants. Better understanding of the relationships between plant nutrients and the host range of herbivorous insects will inform the physiological mechanism by which feeding experience alters host range shifts in A. gossypii.

In this study, we also found that the effect of feeding experience on aphid host range was dependent on aphid genotypes. Feeding experience induced the expansion of host range in some aphid genotypes but not all. Aphids with different genetic backgrounds may have different requirements for nutrition, and consequently live on different host plants. Therefore, the nutrients which can induce changes in host range for different genotypes may be different. It has been found that feeding experience of two clones of the pea aphid, Acyrthosiphon pisum, on alfalfa or red clover did not alter aphid’s specialization to the natal host plant (Via, 1991), and some genotypes of Rhopalosiphum maidis could withstand wheat stressing conditions, but the conditioning on wheat did not decrease the capacity to use their native host Johnson grass (Caballero, Ramirez & Niemeyer, 2001). Genetic differentiations may contribute to host shifts of insects. The rapid adaptation of seed beetle Callosobruchus maculatus to a poor host was mediated by genetic variation at multiple genetic loci, and the contribution of genetic drift was small (Gompert & Messina, 2016). Genetic trade-offs associated with host use have been found in phytophagous insects (Hawthorne & Via, 2001; Gompert & Messina, 2016). Genetic structures of A. gossypii populations on cotton and cucumber plants were different (Liu, Xu & Lei, 2017). Feeding on a constant set of host plants in the life cycle of aphids may result in the genetic differentiation among different host strains, causing the formation of host specialization and evolution of host plant use. Therefore, using genomics techniques will clarify the molecular mechanisms of evolutionary adaptation of insect herbivores to their host plants.

Epigenetic effects and microbial symbionts cannot be ignored in changes of aphid host range (Kim, Thairu & Hansen, 2016). DNA methylation is a general epigenetic mechanism for organisms to adapt to environmental changes. For example, two biotypes of the Russian wheat aphid, Diuraphis noxia, using different host plants showed different methylation levels (Gong et al., 2012). The high variance in the population size of CU3 conditioned by artificial diet and the alternative host plant appeared on cotton plants, and only some aphid genotypes, but not all, could change their host range via the feeding experience on artificial diet. These results imply that there may be epigenetic effects of aphid host range mediated by feeding experience. On the other hand, insect symbionts may be hidden players in insect–plant interactions (Frago, Dicke & Godfray, 2012). Host plant specialization of the pea aphids was found to be governed by a facultative symbiont, R. insecticola (Tsuchida, Koga & Fukatsu, 2004). Although all the aphid genotypes used in this study were not infected with facultative symbionts, the obligate symbiont B. aphidicola was present. The B. aphidicola can supply nutrients for aphids, such as amino acids (Douglas & Prosser, 1992), and changes in the population size of this symbiont can affect fitness of aphids (Chen, Lai & Kuo, 2009; Vogel & Moran, 2011). Symbiont density in aphids was different when aphids fed on different host plants (Zhang et al., 2016). Insect symbionts contributed to a variety of extended insect host phenotypes (Kim, Thairu & Hansen, 2016). We speculate that different dietary nutrients may affect the changes of aphids in epigenomics or microbial symbionts, and consequently results in the change of aphid host range.

Conclusions

The cotton-melon aphid populations exhibit obvious host specialization to cotton and cucumber plants. The aphids on cotton do not use cucumber, and aphids on cucumber cannot establish populations on cotton. Aphids from both the cotton and cucumber can establish populations on an artificial diet, and the short-term feeding experience on this artificial diet results in host expansion of some genotypes. The host range change of aphids mediated by feeding experience on artificial diet is dependent on aphid genetic, epigenetic, or symbiont backgrounds. Nutrients of host plants may contribute to the formation of host specialization and evolution of host plant use in aphid populations. Host specialization may be easy to form in aphid populations feeding on a constant set of host plants in life cycle.

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