Psyllid rearing and hybridization
The original Kyushu colonies were founded by individuals from collections made in 2004 and 2015 within Kumamoto prefecture on the Japanese island of Kyushu, while the source population of the Hokkaido colony originated from individuals collected in 2007 from the Lake Toya area on the island of Hokkaido (Grevstad et al., 2013). The A. itadori populations at MSU were founded via shipments of 480 and 400 Kyushu (southern population) adults in April and June, respectively, and 600 Hokkaido (northern population) adults in June 2021 from colonies maintained at Oregon State University. An additional 350 Kyushu individuals were shipped from the Phillip Alampi Beneficial Insect Rearing Laboratory (New Jersey Department of Agriculture) in August 2021.
We reared the Kyushu population on Japanese knotweed and the Hokkaido population on giant knotweeds (Grevstad et al. 2013) by placing 100 adults into a 40 x 40 x 60 cm mesh cage (Restcloud, Chengdu, China) containing a single potted knotweed plant for 14 days. The adults emerging over the course of 4-5 weeks were kept for an additional 2 weeks in cages to mate and then the next generation was started as described above. The rearing took place in a laboratory at room temperature (22 °C ± 5, RH 40% ± 10, 16L: 8D); under these conditions, psyllids developed from egg to adult in 35 days.
To create the reciprocal crosses knotweed plants infested by either Kyushu or Hokkaido 5th-instar nymphs were cleared of all adults and monitored every 24 hours. Newly-emerging adults were collected, sorted by sex based on the identification of genitalia (Hall, 2008) and paired with similarly collected virgin individuals of the opposite sex and population. Both reciprocal hybrid crosses, the one using Kyushu females paired with Hokkaido males (FemKYU), and the one using Hokkaido females paired with Kyushu males (FemHOK) were reared on Bohemian knotweeds by placing 100 psyllids (50♀ x 50♂) in a cage containing a potted knotweed plant. Adults were kept in the cage for 2 weeks to mate and lay eggs and then removed. Developing psyllids were kept together in the same cage for 8 more weeks that allowed for adult emergence, sexual maturation, and mating. These first-generation (F1) adults were used to start the second generation (F2) without any additional crossing or back-crossing.