Larval instar-dependent hemocytes in specialist herbivore Cydalima perspectalis fed on Buxus hyrcana and Buxus microphylla

The differential interfere contrast (DIC) and light microscopy were used to determine the total (THC) and differential hemocyte count (DHC) in third and sixth instar larvae of Cydalima perspectalis Walker on two boxwood species (Buxus hyrcana and Buxus microphylla). A Neubauer hemocytometer (HBG, Germany) was utilized for THC. The larval hemolymph samples (20 μl) were immediately mixed with 60 μl of anticoagulant solution (0.041 M Citric acid, 0.017 M EDTA, 0.186 M NaCl, 0.098 M NaOH, and pH 4.5) (Amaral et al., 2010). Assays were carried out on larval hemolymph samples individually collected from five larvae (third and sixth instar larvae as replicates) reared on two boxwood species (as treatments). For differential hemocyte counts (DHC), the larvae were submerged in hot distilled water (60°C) for 5 min and then dried with blotting paper. Their hemolymph (20 μl) was collected through a capillary tube from the metathoracic legs of third and sixth instar larvae (< 48 h post ecdysis) fed on two boxwood leaves. According to Gupta (1979), the cell shape, cytoplasmic ratio, cytoplasmic inclusions, and nucleus shape were used for hemocyte identification. The morphometric sizes (length and width) of hemocytes in the third and sixth instar larvae on two boxwood species were determined. In addition, data from total hemocyte (THC) and different hemocyte count (DHC) of two different larval instars fed on the same boxwood species were analyzed using t-test at p < 0.05. Furthermore, total (THC) and different hemocyte counts (DHC) of the same larval instars fed on two boxwood species (B. hyrcana and B. microphylla) were also compared using t-test.