Insect material and host specialization test
We collected A. gossypii from hibiscus (Hibiscus syriacus ) (HI) and cucumber (Cucumis sativus ) (CU) from Baoding (38°53′ N; 115.28′ E) in north China in 2015. The HI and CU lineages were identified using COI sequence and were reared in nylon net cages using cotton and cucumber plants, respectively, under 22±2°C and 16L:8D photoperiod.
Host specialization of A. gossypii was tested with detached leaf (7-d-old) that was placed back side up in 12-cm petri dishes filled with 30 ml 1.5% agar gel. Leaf petiole was wrapped with wet absorbent cotton. The method could keep the leaves fresh for more than 20 d. Five wingless adults were introduced into each petri dish, and were removed 12 h later, leaving less than 20 new nymphs in each petri dish. The petri dish was put in transparent nylon net bag (0.16 mm mesh size), and was placed in an artificial climate chamber under 22±2°C and 16L:8D photoperiod. Four treatments were set: the HI and the CU lineageⅹcotton and cucumber. Each treatment had three replicates (3 petri dishes). The new nymphs were observed for survival, development and reproduction every day until they died.
Life table experiments ofthe HI lineage on fresh and pre-infected cucumber
We chose the biotrophic mycoparasite Pseudoperonospora cubensis(cucumber downy mildew) to pre-infect cucumber leaves because the optimum temperature for P. cubensis epidemic (16℃-24℃) highly overlapped that for A. gossypii outbreak (Savory et al. 2010). More importantly, the same as aphid, P. cubensis stole nutrients from living plant tissues. P. cubensis was collected from cucumber in a greenhouse in Wuhan, Hubei province, China. To identify the pathogen, DNA was extracted from the samples and amplified for the partial sequence of 18s rDNA using the primers ITS1 and ITS4. Spores were washed down from leaves with distilled water to make a suspension of 105 spores per ml. The suspension was sprayed evenly on fresh cucumber leaves (7-d-old), and then the leaves were placed in 12-cm petri dishes filled with 1.5% agar gel. The inoculated leaves were incubated at 22±2 °C in constant darkness for 24 h for infection. Symptoms of infection appeared in 6 to 7 d.
We established life tables of the HI lineage on fresh and pre-infected cucumber leaves in three consecutive generations. Five wingless adults of the HI lineage were introduced into fresh or pre-infected cucumber leaf. The adults were removed and three new born nymphs (F1) were left 12 h later. The F1 nymphs were observed for survival, development and reproduction every day until they died to construct the life table of F1. After being counted, the offspring (F2) of F1 were transferred to their respective leaves (fresh or pre-infected) to produce F3. New F3 nymphs were transferred to their respective leaves (fresh or pre-infected) to construct the life table of F3. Some new F3 nymphs from the pre-infection treatment were transferred to fresh cucumber leaves to construct a life table named HI adapted_fre_F1, with the life table of the CU lineage on fresh cucumber (CU_fre_F1) as control. Each life table replicated 50 aphids.
In order to test whether the HI lineage who was cultured on pre-infected cucumber leaves for two generations (HI adapted) had fully adapted to cucumber plant, we transferred it to heathy cucumber plant of three-leaf stage in a density of 20 aphids per plant to observer the damage symptom. The HI lineage with no experience on cucumber and the CU lineage were introduced to heathy cucumber plant as negative and positive controls. Population growth and damage symptom were qualitatively recorded after two weeks.