The Cellular Immunological Responses and Developmental Differences between Two Hosts Parasitized by Asecodes hispinarum

This study aims to investigate the developmental interactions of Asecodes hispinarum Bouček on Brontispa longissima Gestro and Octodonta nipae Maulik, as well as the cellular immune responses of B. longissima and O. nipae larvae in response to parasitism by A. hispinarum, with the hope of determining the reason for the difference in larval breeding of A. hispinarum in B. longissima and O. nipae. The effects of parasitism by A. hispinarum on the larval development, hemocyte count, and proportion of the hemocyte composition of the two hosts were carried out through selective assay and non-selective assay using statistical analysis and anatomical imaging. There was no significant difference in parasitic selection for A. hispinarum on the larvae of these two beetles; however, more eggs were laid to B. longissima larvae than to O. nipae larvae after parasitism by A. hispinarum. The eggs of A. hispinarum were able to grow and develop normally inside the larvae of B. longissima, and the parasitism caused the larvae of B. longissima become rigid within 7 d, with a high larval mortality rate of 98.88%. In contrast, the eggs of A. hispinarum were not able to develop normally inside the O. nipae larvae, with a high encapsulation rate of 99.05%. In addition, the parasitism by A. hispinarum caused a 15.31% mortality rate in O. nipae larvae and prolonged the larval stage by 5 d and the pupal stage by 1 d. The number of hemocytes during the 12, 24, 48, 72, and 96 h of the four instars from O. nipae larvae was 6.08 times higher than from B. longissima larvae of the same age. After 24 h of being parasitized by A. hispinarum, the total number of hemocytes and granulocyte proportion of B. longissima larvae increased significantly. However, the total number of hemocytes and plasmatocyte proportion of O. nipae increased significantly after 24, 72, and 96 h, and the proportion of granulocytes increased significantly after 12 h post-parasitism. The results in the present study indicated that A. hispinarum was unable to successfully reproduce offspring in O. nipae, but its spawning behavior had an adverse effect on the larval development of its host. In addition, the adequate number of hemocytes and more pronounced changes in the hemocyte count and hemocyte composition ratio in the larvae after parasitization may be important factors for the successful encapsulation in O. nipae larvae.


Introduction
The coconut leaf beetle, Brontispa longissima Gestro (Coleoptera: Chrysomelidae), is thought to be native to Indonesia and Papua New Guinea. The nipa palm hispid beetle, Octodonta nipae Maulik (Coleoptera: Chrysomelidae), is native to Malaysia. These species and the parasitic wasps were fed cotton dipped in 10% sucrose in plastic boxes. After 24 h, the parasitized larvae were dissected. The average parasitism rate and fecundity per female were obtained by counting the number of parasitized larvae and eggs per larva. The experiment was repeated in 40 groups, but only 10 groups had the A. hispinarum eggs counted.
O. nipae used the same experimental procedure as B. longissima.

Selective Parasitism
In the same plastic box, thirty 4th instar larvae of B. longissima and thirty 4th instar larvae of O. nipae were chosen and fed on fresh P. canariensis leaves, respectively. Sixty mated one-day-old female A. hispinarum were introduced to the parasitoid wasps after 0.5 days of upbringing, and the parasitic wasps were fed cotton dipped in 10% sucrose in plastic containers. After 24 h, the parasitized larvae were dissected. The average parasitism rate and fecundity per female were obtained by counting the number of parasitized larvae and eggs per larva. The experiment was repeated in 40 groups, but only 10 groups had the A. hispinarum eggs counted. B. longissima larvae in their fourth instar were selected, placed in separate 9 cm petri dishes, and parasitized by fifteen A. hispinarum mating females in each dish. We dissected B. longissima larvae that were infected by A. hispinarum, using a differential interference microscope. We also observed their progress and captured images at 12,24,48,72, and 96 h after the two insects lay their eggs. Each time point involved the repetition of three groups. The same experimental protocol as B. longissima was followed by O. nipae.
The eggs were stained with rhodamine phalloidin and DAPI and photographed using a differential interference microscope within 24 h. These are the precise steps: The eggs were transferred into a slide coated with poly-L-lysine after 10 µL of PBS was poured upon it. Remove the liquid, absorb 10 µL of PBS rinse, repeat three times, add 10 µL of 4% paraformaldehyde, and then place the wet box in a fixed-temperature environment for 15 min. Rinse three times with 10 µL of PBS, then add 10 µL of 0.1% Triton X-100 and incubate for five minutes in a moist box. Following three rinses with 10 µL of PBS, 10 µL of 1% BSA was added to the wet box for one hour. After three rinses with 10 µL PBS, 10 µL Rhodamine phalloidin and 5 µL (1 µg/µL) DAPI were added and stained in a wet box for 45 min. Rinse three times with 10 µL PBS, then add another 10 µL PBS, cover with a coverslip, and photograph with a differential interference microscope. and placed in separate 9 cm petri dishes. One-day-old, mated female A. hispinarum parasitized each larva one at a time. A. hispinarum was removed 24 h later. The parasitized larvae were constantly fed until they either pupated or became stiff. To count the number of larval deaths, B. longissima and O. nipae fourth instar larvae that were not parasitized throughout the same period were utilized as controls. The experiment was carried out in 30 groups. Ten fourth-instar O. nipae larvae were chosen, and 30 mated female A. hispinarum were inserted to perform one-for-one parasitism. The larvae were used as a control, and the pupal and fourth instar larvae of the palmar anise were counted. The experiment was carried out in thirty groups.

Comparison of the Encapsulation Rates of A. hispinarum Eggs with B. longissima and O. nipae Larvae
Thirty B. longissima 4th instar larvae and thirty O. nipae 4th instar larvae were dissected, and the total number of eggs in each larva and the number of eggs encapsulated were counted. The hemolymph of B. longissima fourth instar larvae was collected at 12, 24, 48, 72, and 96 h after parasitization, and the hemolymph of non-parasitic B. longissima larvae was used as a control at each time. The hemocytes were counted with a blood cell counter (25 × 16) under an optical microscope, including the number of cells and blood cell types; this was repeated five times. At each time point, 30 larvae were taken as replicates, and the experimental method was the same for O. nipae larvae.

Satistical Analysis
The mortality of of B. longissima and O. nipae larvae under non-parasitization and parasitization, the selective parasitism rate of A. hispinarum to B. longissima and O. nipae larvae, and the egg encapsulation rate of these two hosts after parasitization were analyzed using the Chi-square test. The fecundity of B. longissima and O. nipae larvae after parasitization, the duration of the larvae's fourth instar and pupal stage, the hemocyte count, and the proportion of different hemocyte types under parasitization and non-parasitization were compared using an independent sample t-test. The changes in the proportion of hemocyte types at different time points post-parasitization and non-parasitization were analyzed using ANOVA. All statistical analysis mentioned above were performed on SPSS 21, and the graphs were drawn using GraphPad Prism 7.

Parasitic Selection of A. hispinarum to B. longissima and O. nipae Larvae
There was no difference in the parasitism selection of A. hispinarum to B. longissima and O. nipae larvae in the non-selective parasitism experiment (p = 0.859), although there was a significant difference in the number of eggs laid (p < 0.001). The number of eggs produced in the B. longissima larvae was more than that in the O. nipae larvae (Table 1). There was no difference in the parasitism selection of A. hispinarum to B. longissima and O. nipae larvae in the selective parasitism experiment (p = 0.568). In terms of oviposition, there was a significant difference in the quantity of eggs laid (p < 0.001). The quantity of eggs deposited in the B. longissima larvae was more than that in the O. nipae larvae (Table 1).  It can be seen that the eggs could develop normally in the B. longissima larvae ( Figure  2B), and they formed a blastoderm within 24 h, after staining to observe the eggs in fallopian tubes of A. hispinarum and those laid in B. longissima and O. nipae larvae 24 h later ( Figure 2A) (Figure 2B Phalloidin). The eggs laid in the larvae of O. nipae can also develop, but they asphyxiate until they die, because the egg surface is covered in thick hemocytes, in contrast to the eggs in the fallopian tubes of A. hispinarum. ( Figure 2C).   A. hispinarum parasitized B. longissima larva, leaving behind black spots on the body surface where the parasites lay their eggs. The parasitized B. longissima larva gradually turned black over a period of seven days (see Figure 3: the red arrow is where the parasite wasp spawned). O. nipae larva showed localized blackening around the spawning position after being parasitized by A. hispinarum (Figure 4, red arrow), and the eggs enclosed in the larva could be seen through the body surface ( Figure 4, blue arrow). The parasitized larvae can still be observed with the encapsulated eggs until they become adults after extended observation.        (Table 2). When B. longissima larvae were parasitized by A. hispinarum, their death rate was 98.88% ± 0.33%, which was substantially greater than the unparasitized rate of (1.11% ± 0.33%) (χ 2 = 1716.890, df = 1, p < 0.001). O. nipae larvae infected with A. hispinarum had a mortality rate of 15.31% ± 1.62% (Table 2), which was significantly greater than the rate of uninfected larvae (1.44% ± 0.35%) (χ 2 = 112.768, df = 1, p < 0.001). The death rates of unparasitized B. longissima and O. nipae larvae are identical (χ 2 = 0.184, df = 1, p = 0.668). The death rate of B. longissima larvae was considerably higher than that of O. nipae larvae following parasitism by A. hispinarum (χ 2 = 1275.909, df = 1, merged DAPI, merged phalloidin DAPIDAPIABC8 p < 0.001). The statistics of the larval and pupal stages of O. nipae larvae surviving after being parasitized by A. hispinarum (Table 3) showed that the fourth instar larval stage is 12.55 ± 0.09 d, which was 5 d longer than the unparasitized one (7.17 ± 0.08 d); the pupal stage of parasitized was 10.64 ± 0.08 d, which was 1 d longer than the unparasitized one (8.96 ± 0.06 d). O. nipae larval hemocytes promptly encapsulated the A. hispinarum eggs when they were placed in B. longissima and O. nipae larvae. The encapsulation rate reached 99.05%, which was substantially greater than that of B. longissima larval hemocytes (1.14%) (χ 2 = 1352.977, df = 1, p < 0.001) ( Figure 5). Therefore, it stands to reason that eggs cannot survive in O. nipae larvae. The number of hemocytes in the fourth instar larva of B. longissima and O. nipae was maintained at a largely constant level at 12, 24, 48, 72, and 96 h after feeding ( Table 4). The statistics show that O. nipae larvae in their fourth instar have 6.08 more hemocytes than B. longissima larvae (t = −21.496, df = 292, p < 0.001). Eggs O. nipae larval hemocytes promptly encapsulated the A. hispinarum eggs when they were placed in B. longissima and O. nipae larvae. The encapsulation rate reached 99.05%, which was substantially greater than that of B. longissima larval hemocytes (1.14%) (χ 2 = 1352.977, df = 1, p < 0.001) ( Figure 5). Therefore, it stands to reason that eggs cannot survive in O. nipae larvae.

Discussion
In nature, parasitoid wasps and hosts have a balanced struggle and restraint interaction [35,36]. Researchers from all around the world have been investigating the immunological and developmental interactions between parasitoids and hosts in recent decades [37][38][39][40][41]. Depending on whether the parasitoid can develop and enclose successfully in the host, the parasitoid's host can be classified as adaptive or non-adaptive [42]. In contrast to non-adaptive hosts, in which the host is unable to overcome the host's immune system, adaptable hosts allow the host to suppress or avoid the immune system. During the parasitic process, the host must first contend with cellular immunity, with the host hemocytes' envelope reaction serving as the initial hurdle [16,18].
Studies have been conducted on the selection of A. hispinarum as a parasite, the development interaction between A. hispinarum and the hosts B. longissima and O. nipae, and the hemocyte response of these hosts. The findings demonstrated that A. hispinarum exhibited no preference for B. longissima or O. nipae parasitic selection, but that it preferred to deposit more eggs in B. longissima larvae. This might be connected to how big B. longissima and O. nipae are physically. The body size of the B. longissima is larger than that of the O. nipae in all life stages, including larvae, pupa, and adults. During the parasitism process, parasitic wasps typically choose the most suitable host in which to lay eggs and tend to lay more eggs into the most suitable hosts [43]. Host density, body size, nutritional status, and developmental stage are the main influencing factors [44][45][46][47].
However, the eggs laid by A. hispinarum into its adaptive host B. longissima larvae could develop normally, whereas the eggs laid into its non-adaptive host O. nipae larvae were encapsulated to death and could not develop normally. In fact, in the O. nipae larvae, there are some A. hispinarum eggs that can develop into A. hispinarum, but this phenomenon is extremely rare; the current experiment found only one case. Non-adaptive hosts will still be affected by this process, even though parasitic wasps cannot successfully parasitize them. The parasitism of A. hispinarum in this experiment can cause a death rate of 15.31% of the O. nipae larvae and extend the larval stage by 5 days and the pupal stage by 1 day. O. nipae damages the young leaves of palm trees by feeding adults and larvae [48]. Field chemical control often uses beta-cypermethrin, imidacloprid, and other drugs to spray leaves [49,50]. Thus, the extension of the larval stage may allow the insect to absorb the drug more fully, thereby achieving a good control effect. Additionally, we discovered through ongoing field research that though B. longissima and O. nipae can coexist in one area, they rarely feed on the same plants. As a result, utilizing A. hispinarum to regulate B. longissima can have some negative consequences on O. nipae.
The encapsulation response of the host is the first thing the A. hispinarum eggs encounter after being deposited into the host. In this experiment, hemolymph from B. longissima larvae could only encapsulate 1.14% of A. hispinarum eggs, but hemolymph from O. nipae larvae could contain 99.05% of A. hispinarum eggs. Studies have shown a strong correlation between the overall number of host hemocytes and the number of differential hemocytes and the success of parasitic wasps [51]. Statistics show that O. nipae larvae had 6.08 times as many total hemocytes than B. longissima larvae. The amount of circulating hemocytes in the host hemolymph may have a significant role in controlling the capacity to trigger cyst response [52].
The immune system of the host is also suppressed or avoided by parasitic wasps, ensuring the normal development of the offspring [22,29,30]. The hemocytes in the B. longissima larvae did not react significantly to the attack by A. hispinarum, and only a small increase in the total number of hemocytes and an increase in the proportion of granulocytes and oenocytoids were visible in the early stages of the parasitization. This demonstrates that the cell's immune system can act quickly, but it may be slowed down by A. hispinarum's parasitic elements or rendered ineffective by the parasite's eggs, which prevent the hemocytes from encasing the foreign substance. However, after O. nipae larvae were attacked by A. hispinarum, the total number of hemocytes grew, and the proportion of plasma and granular hemocytes increased, which formed the basis for the success of O. nipae larvae's cellular immunity. According to research, the capacity of hemocytes is positively correlated with the number of circulating hemocytes in Drosophila melanogaster [20]. Plasmatocytes and granulocytes are key players in the encapsulation reaction, and oenocytoid cells can produce phenoloxidase to help blacken and kill bee eggs. Additionally, the original hemocytes can change into different types of hemocytes. Since the likelihood of A. hispinarum eggs being encapsulated by the two hosts differ significantly, it may be concluded that A. hispinarum eggs are capable of passive escape, which can only happen in B. longissima larvae.

Conclusions
The goal of this research was to look into A. hispinarum's developmental interactions with B. longissima and O. nipae, as well as the hemocyte immune responses of B. longissima and O. nipae larvae to A. hispinarum parasitism, so that the breeding differences between B. longissima and O. nipae could be explained. The results showed that A. hispinarum had a preference for oviposition over the parasitism of B. longissima and O. nipae. The number of eggs laid to B. longissima larvae was significantly greater than that to O. nipae larvae, which led to a 98.95% mortality rate for B. longissima larvae and only a 15.31% mortality rate for O. nipae larvae. Another important finding is that the sufficient amount of hemocytes in O. nipae larvae served as the foundation for cellular immunity; the number of hemocytes in O. nipae larvae was 6.08 times that of B. longissima larvae. This study's limitation is that it does not take humoral immunity into consideration. The fact that melanization takes place during A. hispinarum egg encapsulation suggests that parasitism will also cause variations in terms of how humoral immunity responds. As a result, we may further investigate various immunological effects on the two beetles from the perspective of humoral immunity.