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

Abstract

The boxwood moth, Cydalima perspectalis Walker (Lepidoptera: Crambidae), as invasive specialist species in Iran has caused considerable damage in endemic forest stands of Buxus hyrcana Pojark since 2016. Host plant species can alter herbivore–plant interactions through the quantitative and qualitative changes of hemocytes even within a specialist herbivore. To determine the hemocyte variations on different host plants across larval development, the third and sixth instar larvae of C. perspectalis fed on B. hyrcana (native boxwood) and B. microphylla Sieb. and Zucc. (introduced nonnative boxwood) were compared. Total (THC) and differential (DHC) hemocyte count were determined using light, phase-contrast, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Based on results, six types of hemocytes were recognized in hemolymph including prohemocytes (PRs), plasmatocytes (PLs), granulocytes (GRs), oenocytoides (OEs) and spherulocytes (SPs) as well as unknown elongated quadrangular cells (elongocytes [ELs], the term has been first employed here) with obvious and distinguishable nuclei which were observed by the SEM and TEM microscopy. The ELs were rectangular or trapezoidal in shape, and the largest cells in hemolymph—their length varied from 14.00 to 15.73 μm. Our results showed that the total number of hemocytes (THC) significantly increased across larval development. Moreover, host plant species significantly affected total hemocyte count where the THC of sixth instar larvae on B. microphylla (2561.00 ± 10.60 cell/mm³) was significantly higher than on B. hyrcana (2440.00 ± 51.50 cell/mm³). The differential hemocyte count (DHC) profile study showed that GRs along with PLs were the most abundant cells in the hemolymph irrespective of larval instar and host plant species. Furthermore, the GRs% and ELs% increased throughout the larval development on both host plants, while a significant reduction of PLs% was recorded from third instar to sixth instar during the larval stage on two Buxus species. Apart from larval instar, host plant species had a significant effect on DHC of C. perspectalis. Despite higher total hemocyte number when fed on B. microphylla, the PLs% and ELs% were significantly higher in sixth-instar larvae fed on B. hyrcana compared with B. microphylla. Oppositely, the percentage of GRs was 17% less in larvae reared on B. hyrcana than on B. microphylla. As hemocyte types are responsible for different immune functions, these findings on instar- and host plant-dependent variation in their relative abundance would be critical to understand the immune response of this specialist herbivore.