2.1. Sampling Collection and Microscopic Examination

Samples of live M. alternatus were captured by traps set in the Taihuyuan pine wood nematode epidemic area of Lin’an (30°35′ N, 119°58′ E), Zhejiang Province, China. Samples of Aulaconotus atronotatus were captured from Jinxiu, Guangxi Province (24°11′ N, 109°58′ E), and Apriona germari along with Paraglenea fortunei were captured from Zhejiang Normal University, Jinhua (29°14′ N, 119°64′ E), Zhejiang Province, China. After morphological identification, the specimens of M. alternatus were then cultivated in the laboratory under artificial feeding conditions. Cultivation was in a plastic incubator that was placed in an artificial climate box; fresh Masson pine was provided every 3 days, the temperature in the box was maintained at 20–25 °C and relative humidity was 60–70%. Specimens of the other three longicorn beetles were deposited at −40 °C in the Animal Specimen Museum, College of Life Sciences and Chemistry, Zhejiang Normal University, China. After experimentation, all the pine wood was incinerated.

All M. alternatus were quickly dissected after a week and small pieces of abdominal tissue, thorax flight muscle, and intestinal tract were taken for microscopical examination. We used an OLYMPUS-SZX16 stereo microscope to observe abdominal tissue samples to directly determine the presence/absence of nematodes. If nematodes were found in M. alternatus tissue, then tissues from that individual were assigned to the experimental group whereas beetles without nematodes were placed into the control group. The excised digestive tract and flight muscle tissue samples were quickly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80 °C until use.