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.