How Does Vector Bemisia Tabaci use Visual and Olfactory Cues in Orientation to A Virus-Infected Host Plant?

Cucurbit chlorotic yellows virus (CCYV) has caused serious damage to melon crops in many countries in recent years. It is exclusively transmitted by the notorious pest Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a semi-persistent manner. Previous studies have shown that both persistently and nonpersistently transmitted viruses can manipulate orientation and performance of vector insects through changing host phenotype to facilitate virus spread. However, as a semi-persistently transmitted virus, how CCYV affect vector B. tabaci in locating host plants by altering physiological traits of host plants is still unknown. In this study, we investigated B. tabaci visual and olfactory preference between healthy and CCYV-infected host plant Cucumis sativus. Volatile proles of healthy and CCYV-infected C. sativus plants were analyzed by using gas chromatography-mass spectrometry (GC-MS). Initially, it was found that vector insects preferred to settle down CCYV-infected Cucumis sativus seedlings in free choice. The concentrations of total volatiles and terpenes in cucumber plants were notably decreased after CCYV infection, and especially, the concentrations of α-pinene, β-ocimene, α-farnesene, and nonanal, responsible for olfactory attraction of B. tabaci, dramatically reduced in CCYV-infected plants. Subsequently, we investigated the visual preference of B. tabaci to CCYV-infected and healthy host, and found that B. tabaci adults showed signicant preference to CCYV-infected host. CCYV induced yellowing symptoms in host leaves may explain the visual preference of B. tabaci adults to infected hosts. This study indicated that visual cues could play a vital role in vector insects locating virus-infected host plants.


Introduction
More than 80 percent of plant viruses are dependent on vectors for spread, so virus transmission is closely related to behaviors and biological traits of vector insects (Hohn 2007  However, some studies indicated that viruses with various transmission manners induced different plant phytochemical changes. As for non-persistently transmitted viruses, vector insects could acquire virions during short brief ingestion, and virions could be lost with salivary secretion (Ng et al. 2006

Impacts of CCYV on B. tabaci Host Selection Preference
Bi-choice Assay CCYV-infected and healthy C. sativus seedlings were placed alternately in insect cages (60 cm × 60 cm × 60 cm), and 100 viruliferous and non-viruliferous B. tabaci adults, after 30 min starvation, released into the middle area of the cage. The number of B. tabaci adults on each plant were counted at intervals of 1hr, 3hr, 5hr and 24hr. Four replicates were conducted in each group.
Olfactory Preference to Plant Extracts Healthy and CCYV-infected plant leaves were ground into powder with liquid nitrogen. Two grams of plant leaf powder were soaked in 10 mL 80% methanol solution for 24 hours, and the extracts were used for Y-shaped tube test. Standard chemicals (purity > 99%) were diluted with para n oil with para n oil used as control.
Two hundred microliters of plant extracts or diluted chemicals were added onto the 3 × 2 cm lter paper, and the two pieces of lter paper were separately placed into two bottles connected with a Y-shaped tube olfactometer. The olfactometer consist of one main tube and two arms (glass tubes, each with 10 cm long and 2 cm internal diameter) with 60° angle between the two arms. Flow of the odor was blown into the tube at 0.3 L/min for 10 minutes before insects were released into the end of the main arm. An adult was tested each time, and behavioral selection responses were observed. When an insect crossed 1 / 3 of an arm within 3 min, it was regarded as an e cient choice. Twenty adults were tested in each group with 3 replicates.
Collection and Analysis of Plant Volatiles Volatiles from aerial parts of infected and healthy host plants were collected by using aeration sampling system as described by Mwando et al. (2018). The plant was contained in an oven plastic bag (50 cm × 55 cm) and connected to an air-generating device with an inlet ow rate of 0.3L/min. Volatiles were collected by passing the outlet air through a PoraPak Q (60 mg, mesh 50-80, supelco, Bellefonte, PA, USA) lter at the rate of 0.1 L/min. Before use, PoraPak Q lters were washed sequentially with hexane, acetone and diethyl ether for three times, and dried by a stream of nitrogen gas. After 8hr collection, the lters were eluted with 500 µL hexane and then stored at -80 ℃ for analysis. Collection of volatiles were conducted from 10:00 p.m-6:00 a.m. Collections for each group of plants were repeated for ve times.
Collected volatiles samples were analyzed using gas chromatographic-mass spectrometry (GC-MS) (Agilent 7890B couple 5977, Agilent Technologies, USA), equipped with a non-polar HP-5 MS column (30 m × 0.25 mm, 0.25 µm lm). One microliter sample was injected into GC system with helium as a carrier gas at a ow rate of 1.0 ml/min. The temperature program was 40℃ for 2 min, raising up to 180 ℃ at the rate of 5 ℃/min, nally up to 250 ℃ in the rate of 15 ℃/min. Chemical spectra were recorded at 70 eV in the electron impact (EI) ionization mode. Substances were identi ed by comparing with mass spectral data library (NIST 14.0) and Masshunter working station. Typical chemicals were identi ed by comparing the retention time and mass spectrogram with standard chemicals. The standard chemical nonylacetate was diluted with hexane to 6 concentrations, and the external standard curve was established according to the different concentrations and peak areas (R 2 = 0.9976). The concentration of chemicals were calculated by the standard curve.
White y B. tabaci Visual Preference Assay One hundred adult insects, after starved for 30 min, were released into the center of the release point in the glass box (Fig. 1). Plants infected by CCYV for 20 days and healthy plants were placed on both sides of the lower area. Because of the airtight glass plates and tubes, air can't circulate within the device. The number of insects in both tubes were counted after 1 hr. Experiments were repeated seven times. There is no air ow within the device, and we exchanged positions of two groups of plants every time. Thus, the environmental in uence could be eliminated.
Statistical Analysis SPSS 22.0 was used to analyze the differences between the treatment and the control group. The signi cant levels were set as P < 0.05 ('*'), P < 0.01 ('**'), and P < 0.001 ('***'). For data analysis of B. tabaci behavioral response tests, the number of B. tabaci was transferred into a percentage for difference analysis (Tamiru et al. 2011).

Page 6/19
Virus Titers Determination in Plants The virus titers varied signi cantly at different stages. At 10 days after CCYV infection, the amount of coat protein gene molecules was 1.40 × 10 2 copies, was 2.94 × 10 6 copies at 20 days, virus titer was 2.33 × 10 6 copies at 30 days, while nothing was found in healthy cucumber leaves (Fig. 2). With the increase of virulence titers, the yellowing symptoms got more obvious.
B. tabaci bi-choice Assay White y B. tabaci bi-choice assay showed that, after releasing insects for 3 hr, the percentages of B. tabaci selecting CCYV-infected plants were signi cantly higher than that for healthy plants (Fig. 3), and both viruliferous and non-viruliferous B. tabaci adults preferred to settle down CCYVinfected host plants. This result implies the CCYV didn't affect vector's preference to virus-infected plants.
Visual Preference to Infected and Healthy Plant Test In visual selection test, the percentage of B. tabaci selecting CCYV-infected plant was signi cantly higher than that to healthy plants (Fig. 7) (P < 0.05). It indicates CCYV enhanced the visual preference of B. tabaci to CCYV-infected host plants.

Discussion
In this study, we found that CCYV infection improved vector insect B. tabaci the visual cues rather than olfactory cues in orientation to host plants. CCYV decreased volatiles emissions, especially the terpenes. Availability of data and material All data generated or analyzed during this study are included in this published article.   represents the percentage of B. tabaci prefer to healthy hosts in each group. '*' means P < 0.05, '**' means P < 0.01, '***' means P < 0.001. Independent-sample t test was used to analyze the difference (P < 0.05).

Figure 5
Total amounts of volatiles and terpenes analysis from Non-infected and CCYV-infected Cucumis sativus seedlings Columns show Mean ± Standard Error. '*' means P < 0.05, '**' means P < 0.01, independentsample t test was used to calculate the difference. the number of B. tabaci was transferred into percentage for analyzing difference according to independent-sample t test. Three replicates were conducted. '*' means P < 0.05, '**' means P < 0.01, '***' means P < 0.001.

Figure 7
Visual selection preference of B. tabaci between CCYV-infected and healthy plants Data = Mean ± Standard Error. One hundred adult insects were tested and seven replicates were carried out. The number of B. tabaci was transferred into percentage for analyzing difference by independent-sample t test ('*' < 0.05).