A Semipersistent Plant Virus Differentially Manipulates Feeding Behaviors of Different Sexes and Biotypes of Its Whitefly Vector

It is known that plant viruses can change the performance of their vectors. However, there have been no reports on whether or how a semipersistent plant virus manipulates the feeding behaviors of its whitefly vectors. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emergent plant virus in many Asian countries and is transmitted specifically by B and Q biotypes of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. In the present study, we used electrical penetration graph (EPG) technique to investigate the effect of CCYV on the feeding behaviors of B. tabaci. The results showed that CCYV altered feeding behaviors of both biotypes and sexes of B. tabaci with different degrees. CCYV had stronger effects on feeding behaviors of Q biotype than those of B biotype, by increasing duration of phloem salivation and sap ingestion, and could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, with more phloem ingestion in Q biotype males and more non-phloem probing in B biotype males than their respective females. With regard to feeding behaviors related to virus transmission, these results indicated that, when carrying CCYV, B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females.


Introduction
Most plant viruses depend on vector insects to move from one host to another and over distantly-located regions [1][2][3][4]. The interaction between virus and vector is very specific and complex, and some studies reported that viruses could alter directly the physiology and behaviors of the vector to promote spread of the viruses [5][6][7][8][9][10][11]. The implications of the interactions among virus, vectors and plants in virus pandemics have attracted more and more attention in recent years [12,13].
To obtain CCYV-infected plant cultures, cucumber plants at 1-2 true-leaf stage were inoculated with Agrobacterium tumefaciens-mediated CCYV clones [41]. Plants were then left to grow for 30 days, and infection status was determined by symptom of chlorotic leaf spots and yellowing, and subsequently confirmed by real-time RT-PCR. All plants were maintained in separate insect-proof cages under a greenhouse under the above conditions.

Laboratory Whitefly Populations
The whitefly populations of B. tabaci B and Q biotypes were reared on healthy tobacco (Nicotiana tabacum cv. Zhongyan-100) plants for many years in whitefly-proof screen cages (60 cm × 40 cm × 80 cm) under conditions as above, respectively. The purities of B. tabaci B and Q biotype populations were monitored every 4-5 generations by using the mitochondrial cytochrome oxidase I (mtCOI) genes [42,43].

Establishment of Non-Viruliferous and Viruliferous B. Tabaci Populations
We established non-viruliferous and viruliferous whitefly colonies by transferring ca. 600 adults of B. tabaci B and Q biotypes from above laboratory populations into each cage with two virus-free or CCYV-infected cucumber plants, respectively. Viruliferous and non-viruliferous whitefly colonies were maintained for 2 generations in a greenhouse under above conditions. Starting from the third generation, we randomly selected newly emerged whiteflies (2 to 5 day-old males and females) from each colony for use in the experiments.
Total RNA of the individual adult whitefly from CCYV-infected or non-infected plants was extracted using TRlzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. RNA concentration and purity were measured in a NanoDrop TM spectrophotomer (Thermo Scientific, Wilmington, DE, USA) and stored at −20 • C for subsequent analysis. Total RNA (1 µg) from each sample was reverse transcribed to generate the first-strand cDNA using the PrimeScript ® RT reagent Kit (Takara, Dalian, China).

Electrical Penetration Graph Recording
A direct-current EPG (DC-EPG, Model Giga-4) system (Wageningen University, Wageningen, The Netherlands) was used to monitor the feeding behaviors of viruliferous and non-viruliferous whitefly adults on cotton plants. We used cotton plants in the experiments because cotton is the excellent host plant for whiteflies, but not the host of CCYV, to avoid virus effects on non-viruliferous whiteflies that could mask the direct effects of the virus on its vector. Whiteflies were immobilized on the ice pack, then we attached a gold wire (1.5 cm × 12.5 µm) to the pronotum of a whitefly using a drop of water-based silver glue. The wired whiteflies were starved for ca. 20 min before connected to the Giga-4 probe input and placed onto the lower surface of the first true leaf. Six hours of EPGs were continuously recorded for each replicate with a fresh adult and a new plant. All experiments were carried out in a quiet room at 26 ± 1 • C, at 75% ± 0.5% relative humidity and under 1000 lux artificial light. All the recoding experiments were finished in an electrically grounded Faraday cage to shield against external electrical noise.
The EPG signals were digitized with a DI-720-UL analogue-to-digital converter (Dataq Instruments, Akron, OH, USA), and the output was acquired and stored with Stylet+ for Windows software (Wageningen University, Wageningen, The Netherlands), and data were analyzed with this software after data conversion.

Data Analysis
Waveforms patterns were categorized as previously described [44][45][46]. We identified five distinct waveforms in this study: non-probing (np; non-probing behavior); pathway [C; intercellular stylet pathway, including, if occurred, waveforms F (presumed penetration difficulties) and G (xylem sap ingestion)]; potential drop (pd; puncturing into the tissue cells and tasting the cytoplasm); the phloem phase E1 (watery saliva secretion into a sieve element) and E2 (ingestion of sieve element sap). The time from the start to the end of each waveform was recorded and exported by using Stylet+ software.
We selected 16 EPG variables (8 non-phloem phase variables and 8 phloem phase variables) for analysis and comparison of feeding behaviors of different biotypes and different sexes of B. tabaci with or without CCVY. All statistical analyses were done with IBM SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA) and significant differences were tested at the 0.05 or 0.01 level. Prior to analysis, normality and homogeneity of variance were checked. Data were log10 transformed when it did not fit a normal distribution. Means of viruliferous and non-viruliferous whiteflies of the same sex and the same viruliferous situation were compared by Mann-Whitney test. Two-way analysis of variance (ANOVAs) was used to analyze the interaction effects of sex and whitefly viruliferous status (viruliferous and non-viruliferous whitefly). Multivariate analysis of variance was used to analyze the interaction effects of biotype (B and Q), sex (male and female), whitefly viruliferous status (non-viruliferous and viruliferous). Means were compared by least significant difference (Tukey's) tests.

Results
A total of 166 successful EPG recordings were obtained on cotton plants, a non-host of CCYV, including 73 for B. tabaci B biotype whiteflies (37 non-viruliferous whiteflies with 18 replicates for males and 19 replicates for females, and 36 viruliferous with 17 replicates for males and 19 replicates for females), and 93 for Q biotype whiteflies (50 non-viruliferous with 20 replicates for males and 30 replicates for females, and 43 viruliferous with 22 replicates for males and 21 replicates for females).

Effects of CCYV on Phloem Feeding Behaviors of B. tabaci B and Q Biotypes
From 8 phloem EPG variables selected (Figure 2), we found that CCYV stimulated phloem salivation and sap ingestion in both biotypes of B. tabaci, with stronger effects on feeding behaviors of biotype Q than those of biotype B. Viruliferous B and Q biotype whiteflies had a highly significantly greater total number of E2 (for B biotype, 2.03 ± 0.  Data are means ± SE; bars with asterisk(s) (* or **) indicate a statistically significant difference between the non-viruliferous and viruliferous whiteflies of same biotype at p < 0.05 or p < 0.01 (Mann-Whitney test). EPG waveforms: C = pathway; np = non-probing/penetration; pd = potential drop (intracellular puncture); E = phloem salivary secretion and/or sap ingestion.

Effects of CCYV on Feeding Behaviors of the Different Sexes of B. tabaci B and Q Biotypes
The effects of CCYV on feeding behaviors of the different sex of non-viruliferous and viruliferous B and Q biotypes whiteflies were shown in Tables 1 and 2.

Feeding Behaviors of Male Whiteflies
Non-phloem EPG variables (Table 1). Compared to non-viruliferous B and Q biotypes male whiteflies, viruliferous B and Q biotypes male whiteflies had significantly shorter time to first E from first probe and time to first E ( Table 1, Variables F and G). Furthermore, viruliferous B and Q biotypes male whiteflies spent significantly less total duration of np (Table 1, Variable D), and viruliferous B male whiteflies had a significantly greater total number of pd (Table 1, Variable E). No significant differences were detected between non-viruliferous and viruliferous males or females in both biotypes in the duration of first probe, total number of C, total duration of C (Table 1, Variables A, B and C).
Phloem EPG variables ( Table 2). Viruliferous B and Q biotypes male whiteflies had a significantly greater total number of E2 and total duration of E2 (Table 2, Variables L and M) than non-viruliferous male whiteflies. Furthermore, viruliferous Q biotypes male whiteflies had a significantly greater total number of E1, total duration of E1, total number of E1 after first E2, total duration of E1 after first E2, and percentage of E (Table 2, Variables J, K, N, O and P), but non-viruliferous and viruliferous B biotypes male whiteflies did not differ in these variables. Total duration of first E1 did not differ between non-viruliferous and viruliferous males in both biotypes ( Table 2, Variable I).

Feeding Behaviors of the Female Whiteflies
Non-phloem EPG variables (Table 1). Viruliferous Q biotype female whiteflies had significantly shorter time to first E from first probe and time to first E ( Table 1, Variables F and G), and a significantly greater total number of pd (Table 1, Variable E) than non-viruliferous Q biotype female whiteflies. Moreover, viruliferous B biotype female whiteflies had significantly shorter total number of C (Table 1, Variable B) than non-viruliferous B biotype female whiteflies. Other non-phloem variables, including duration of first probe, total duration of C, total duration of np, total number of probes before first E, did not differ between non-viruliferous and viruliferous females in both biotypes (Table 1, Variables A, C, D and G).
Phloem EPG variables (Table 2). Viruliferous Q biotype female whiteflies had significantly shorter total duration of first E1 than non-viruliferous Q biotype female whiteflies ( Table 2, Variable I). Compared to non-viruliferous Q biotyoe female whiteflies, however, viruliferous Q biotype female whiteflies had more total duration of E2 and higher percentage of E ( Table 2, Variables M and P), but non-viruliferous and viruliferous B biotypes female whiteflies did not differ in these variables. Other phloem variables, including total number of E1, total duration of E1, total number of E2, total number of E1 after first E2 and total duration of E1 after first E2, did not differ between non-viruliferous and viruliferous females in both biotypes (Table 2, Variables J, K, L, N and O).

Comparison of Feeding Behaviors of the Male and Female Whiteflies between Biotypes
Non-phloem EPG variables (Table 1). Non-viruliferous B biotype male and female whiteflies had a greater total number of C and total number of probes before first E than non-viruliferous Q biotype male and female whiteflies (

Interaction Effects of CCYV, Sexes and Biotypes
Two-Way ANOVA Analyses Table 3 presents the two-way ANOVA statistics on feeding behaviors of the different sexes with non-viruliferous and viruliferous B. tabaci B or Q biotype, respectively.
(i) EPG variables of B biotype whiteflies. The sex of whitefly had significant effects on 3 out of 16 variables examined, i.e., the total duration of np, total number of pd and total duration of E2 (Table 3, Variables D, E and M). Whitefly viruliferous status appeared to exert significant effect on 5 of 16 variables, including total number of pd, time to first E from first probe, time to first E, total number of E2, total duration of E2, total number of E1 after first E2 (Table 3, Variables E, F, H, L and N). The interaction of the two variables exerted a significant effect on the total duration of np, total number of pd, time to first E from first probe, time to first E, total duration of E2 (Table 3, Variables D, E, F, H and M). (ii) EPG variables of Q biotype whiteflies. The sex of whitefly appeared to exert significant effect on 5 out of 16 variables, i.e., the total duration of C, total duration of np, total number of E1, total number of E2, total number of E1 after first E2 (Table 3, Variables C, D, J, L and N). The viruliferous status exerted significant effects on 10 variables, including total number of pd, time to first E from first probe, time to first E, total number of E1, total duration of E1, total number of E2, total duration of E2, total number of E1 after first E2, total duration of E1 after first E2, and percentage of E (Table 3, Variables E, F, H, J, K, L, M, N, O and P). The interaction of the two variables showed a significant effect on the total number of E2, total duration of E2, and total duration of E1 after first E2 (Table 3, Variables L, M and O). It clearly indicated that the whitefly viruliferous status exerted remarkably stronger effects on whitefly feeding behaviors than the sex on cotton plants, and viruliferous status had stronger effects on Q biotype than B biotype. Table 4 presents the multivariate statistics on feeding behaviors of the different sex with non-viruliferous and viruliferous B. tabaci B and Q biotypes. The biotype of whitefly had significant effects on 3 out of 16 variables, i.e., the total duration of C, total number of pd, and total number of probes before first E (Table 4, Variables B, E and G). The sex of whitefly had significant effects on 4 out of 16 variables, including the total duration of C, total duration of np, total number of pd and total duration of E2 (Table 4, Variables C, D, E and M). Whitefly viruliferous status showed significant effect on 4 non-phloem variables (the total number of pd, time to first E from first probe, total number of probes before first E, time to first E) and 5 phloem variables (total number of E1, total number of E2, total duration of E2, total number of E1 after first E2 and percentage of E) ( Table 4, Variables E, F, G, H, J, L, M, N and P). Interaction of the biotype and sex exerted a significant effect on the total duration of E2 (Table 4, Variable M), and the interaction of biotype and viruliferous status showed a significant effect on the total number of pd (Table 4, Variable E), while the interaction of sex and viruliferous status appeared to exert significant effect on the total duration of np, total number of pd, total number of E2, total duration of E2 (Table 4, Variable D, E, L and M). The interaction of the biotype, sex and viruliferous status exerted a significant effect on the total number of pd (Table 4, Variable E). It clearly indicated that the whitefly viruliferous status exerted remarkably stronger effects on whitefly feeding behaviors than the sex or biotype on cotton plants.

Discussion
In this study, we found that Cucurbit chlorotic yellows virus (CCYV) could differentially manipulate feeding behaviors of its vector B. tabaci with varying degrees on sexes and biotypes. This is the first report about a semipersistly transmitted virus manipulating feeding behaviors of different sexes and biotypes of its whitefly vector. Importantly, the altered feeding behaviors of viruliferous whiteflies seemed likely to increase the rates of CCYV transmission. Viruliferous whiteflies spent more time salivating into sieve elements than did non-viruliferous whiteflies. Salivation is essential for the virus transmission [5]. More time spent in E1, and a shift toward a larger number of short feeding bouts, should maximize viral inoculation [7].
In the EPG experiments, we used cotton plants because they are excellent host plants for whiteflies and immune to CCYV. This set-up enabled us to eliminate the effects of plant-mediated indirect modifications due to virus infection [47] that could mask the direct effects of the virus on its vector. Here, we found that viruliferous whiteflies showed significantly higher feeding efficiency, and the interactions between plant viruses and their insect vectors are not neutral. The non-viruliferous B and Q biotypes whiteflies, for example, took about 1.5 times more time probing to the phloem of plants than viruliferous whiteflies ( Figure 1F,H), while viruliferous whiteflies had about 1.4 times more in the total number of E2 ( Figure 2L) and about 1.5 times greater total number of E1 after first E2 ( Figure 2N) than non-viruliferous whiteflies. Similarly, compared to non-viruliferous whiteflies, whiteflies carrying Tomato yellow leaf curl virus (TYLCV, a persistently transmitted virus) took more salivating time into sieve tube elements and reached to the phloem of plants more quickly [7], and viruliferous whiteflies were more restless and had more attempted probes and salivation [8].
B and Q biotypes whiteflies of B. tabaci differed in several aspects of their feeding behaviors in regard to CCYV effects. Compared to non-viruliferous whiteflies, for example, viruliferous Q biotype whiteflies had a significantly greater total number of E1, total duration of E1, total duration of E2, total duration of E1 after first E2 ( Figure 2J,K,M,O) than viruliferous B biotype whiteflies. The total time spent in sap ingestion ( Figure 2M) was most strongly correlated with viral acquisition, and the total time spent in salivating into sieve elements ( Figure 2J,K,O) was most strongly correlated with viral inoculation [5]. This agrees with the results of the previous study [7] that B. tabaci B and Q biotype whiteflies were affected similarly by the presence of TYLCV, but this does not mean that they are equally effective viral vectors. Our study found that Q biotype whiteflies had a stronger ability to transmit CCYV, the semipersistent virus, than B biotype whiteflies [48].
In B. tabaci and many other piercing-sucking insects, males are smaller in size and more active than females, and faster movements of males may facilitate virus transmission. In the coevolution of plant viruses and their insect vectors, viruses probably rely more on males than females in dispersal, so it can be presumed that feeding behaviors of males may be manipulated greater in males by viruses. In this work, we found that the feeding behaviors of different sexes of viruliferous B and Q biotypes whiteflies were manipulated to different degrees when they were carrying CCYV (Table 3). For example, viruliferous male whiteflies of both biotypes could reach to the phloem of plants more quickly (Table 1, Variables F and H) and spend more time feeding on sap (  [49] reported that the feeding ability of TYLCV-infected whiteflies was significantly affected by the sex. CCYV-infected whitefly males and females had a greatly different performance between B and Q biotypes whiteflies. Similar result was reported in Stafford and collaborators [6] that the feeding behavior of the thrip Frankliniella occidentalis males carrying Tomato spotted wilt virus (TSWV) was modified to enhance virus transmission. Moreover, in this study, the viruliferous B biotype male whiteflies had significant more potential drops (pd), representing puncturing into the tissue cells and tasting the cytoplasm ( Figure 1E; Table 1, Variable E), than non-viruliferous male whiteflies. The intracellular probing is related to the spread of non-persistent viruses [50], and the relationship between the intracellular probing and the spread of phloem-restricted semipersistent virus needs further study.
Manipulation of vector feeding behaviors by the plant virus is an evolved mechanism to facilitate virus transmission [51][52][53][54][55], but mechanism underlying this phenomenon is not clear. A number of internal and external factors, such as the age of the vector, virus quantity within the vector, disease progressing, and the growth stage of the plant, lead to the three-way complex dynamic interactions among viruses, vectors and plants [56]. Some studies had revealed the interactions between a virus and its vectors may vary with the vector and plant species, even with different strains of the same species. TSWV activates the immune system of its vector, the F. occidentalis [57]. Moreover, transcriptome analysis of B. tabaci MEAM1 putative species (Q biotype) with or without Tomato yellow leaf curl China virus (TYLCCNV) showed that TYLCCNV may have direct adverse effects on MEAM1, such as disturbed circulation and metabolism in the cell [58]. Wang and collaborators [59] have studied the Angiotensin-converting enzymes (ACEs) in aphid saliva finding that when ACE1 and ACE2 were simultaneously knocked down, aphid feeding was enhanced, such as aphids required less time to reach the phloem and showed longer passive ingestion. Does this suggest that virus particles binding to vector receptors can change some gene expression and thereby enhance the feeding behaviors of whiteflies or other insect vectors? In addition, whether endosymbionts of the organisms affect the tripartite interactions among viruses, vectors and plants is still barely understood [60,61]. The presence of Hamiltonella was involved in acquisition, retention, and transmission of TYLCV by B. tabaci B and Q biotypes in significant differences for TYLCV accumulation in plants exposed to the whiteflies [62]. CCYV and some other Crinivirus were proven to bind to receptors in the foregut within the whitefly vectors [3,48], but little information is available in molecular mechanisms underlying changes of vectors' feeding behaviors when carrying these semipersistently transmitted viruses.

Conclusions
In conclusion, our current study confirmed that CCYV, a non-circulative phloem restricted Crinivirus, could manipulate the probing and feeding behavior of its vector, B. tabaci, in a manner that facilitates its own transmission. Interestingly, the manipulation degrees vary with sexes and the biotypes of whiteflies. We found that CCYV had more effects on feeding behaviors in Q biotype than in B biotype, by increasing duration of phloem salivation and sap ingestion. CCYV could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, by the order as Q males > B males > Q females > B females, with more phloem ingestion in males than females in Q biotype and more non-phloem probing in males than females in B biotype. These results clearly indicated that the probing and feeding behaviors of B. tabaci B and Q biotypes changed greatly when carrying CCYV, with varying degrees with biotypes and sexes. From these EPG results of feeding behavioral changes of whitefly vectors carrying CCYV, we can presume that B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females in CCVY transmission. Our studies have clear implications for understanding the behavioral mechanisms underlying a mutualistic relationship between an insect vector and a plant virus, and may help to improve management of different B. tabaci biotypes and the semipersistly transmitted virus (CCYV and other Crinivirus virus species).