Pathology Of Matsucoccus Matsumurae And Ceroplastes Japonicus Infected By Entomopathogenic Fungi

Scale insects, in classification, belong to Hemiptera, Coccoidae in Insecta. About 7500 species belonging to 49 families were recorded around the world. They are closely related to aphids in evolution. They are one group of the important economic insects because most species of them are important pests in agricultural crops, forests, fruit trees and flower plants. The special characteristic of the scale insects is that they possess many kinds of wax glands and can secret wax substances to form wax test or wax covering on the body surface. These wax secretions protect the scale insects from the attack of chemical insecticides, so that it is difficult to control the scale insects.In order to develop a new biological control way by using fungal pathogens to infect scale insects and cause them diseased and death to control their populations and damage, two species of the scale insects, Matsucoccus matsumurae (Kuwana) and Ceroplastes japonicus Green were chosen as target insects in this study. The former is the type species of the family of Matsucoccidae and the genus of Matsucoccus, a group of special pests restricted in Pinus spp., and it is also a representative species in Archaeococcoids, and the latter belonging to the family of Coccidae and the genus of Ceroplastes, is a species with a wide distribution, a large number of host, and thick wax test on the dorsal surface, and it is also a representative species in Neococcoids. The entomopathogens Lecanicillium lecanii V3.4504 strain and V3.4505 strain, and Fusarium incarnatum-equiseti HEB01 strain and HEB02 strain, originally isolated from the scale insects and brown planthoppers, were chosen as the test fungal species. The infection process, pathological symptoms and the biochemical responses were investigated in the scale insect infection.The results on the study of Matsucoccus matsumurae are as follows:1. Morphological characteristic, wax secretion characters and development progress of Matsucoccus matsumuraeMatsucoccus matsumurae has a complex metamorphosis, and its morphological characteristics and wax secretion characters varied in different instars. Moreover, Matsucoccus matsumurae distributed from northeast to south along the east region of China, in which, the climate are greatly varied that resulted in the scale insects developed in a different process. However, the information in this aspect is poor, so that it difficult to guide the biocontrol by using pathogens. In the present study, three representative climate zones were chosen at Jinhua in Zhejiang Province (ZJJH), Qingdao in Shandong Province (SDQD) and Fushun in Liaoning Province (LNFS), respectively. The experiment forests were selected in the forests with infestation of M. matsumurae. The investigation in forest and the observation in the laboratory were conducted. The development of instars, morphological changes, wax secretion, and the behavior of post-hibernation, overwintering, mating and laying eggs were studied.The results showed that although M. matsumurae reproduced two generations a year in the three regions, the developmental process was different obviously. In ZJJH, the post-hibernation nymphs emerged in early March, in SDQD, the post-hibernation nymphs emerged in late April-early May, and in LNFS, the post-hibernation nymphs emerged in middle May-late May; whereas, the populations of LNFS began to overwinter in October, but the populations of ZJJH did not overwinter until December. These differences were possibly related to the local temperature. A lot of biological characteristics and rhythm were found, especially, pearl-like morph of the 2nd-instar nymphs and their emergence process in early spring, wax filament secretion and cocoon construction of the male 3rd-instar nymphs, eclosion, courtship and mating of the male adults, emergence, mating, wax filament secretion, egg sac formation and egg deposition of the female adults were observed. These results provided a basis for the pathological research and biological control of M. matsumurae.2. Micro-and ultrastructure of the female reproductive system of Matsucoccus matsumuraeThe adult female of Matsucoccus matsumurae is at a mating and reproductive period and the ovaries occupied most space of its hemocoele. In this stage, the pathological changes of the ovaries must be involved with the infection of the pathogens. However, the structure of the reproductive system of the adult female has never been reported. In the present study, the ultra-and micro structure of the female reproductive system was observed using light microscopy, scanning and transmission electron microscopy to provide basis for the histopathological studies. The results revealed that the female reproductive system of M. matsumurae was composed of a pair of ovaries, a common oviduct, a pair of lateral oviducts, a spermatheca and two pairs of accessory glands. Each ovary is composed of approximately 50 telotrophic ovarioles that are devoid of terminal filaments. Each ovariole is subdivided into an apical tropharium, a vitellarium and a short pedicel connected to a lateral oviduct. The tropharium contains 8-10 trophocytes and two early previtellogenic oocytes termed arrested oocytes. The trophocytes degenerate after egg maturation, and the arrested oocytes are capable of further development. The vitellarium contained 3-6 oocytes of different developmental stages:previtellogenesis, vitellogenesis and choriogenesis. The surface of the vitellarium is rough and composed of a pattern of polygonal reticular formations with a center protuberance. The oocyte possesses numerous yolk spheres and lipid droplets, and is surrounded by a mono-layered follicular epithelium. A binucleate was firstly observed in the follicular cells at the beginning of vitellogenesis and accessory nuclei are observed in the peripheral ooplasm during vitellogenesis. By this research the fine structure of the female reproductive system of M. matsumurae was given, it is the first time in scale insect studies of the world. It provided a new character for the taxonomy in Archaeococcoidea and it is significant for understanding the evolution of M. matsumurae. And it provided important basis for studying the fungal infection and analyzing the pathological symptom of the ovaries.3. Pathogenicity of the entomopathogenic fungi, Lecanicillium lecanii and Fusarium incarnatum-equiseti to Matsucoccus matsumurae The pathogenicity of entomopathogenic fungi, Lecanicillium lecanii strain V3.4504 and V3.4505 and Fusarium incarnatum-equiseti strains HEB01 and HEB02, against M. matsumurae was tested in four instars to evaluate their potential as a biological control agent. The results showed that the four strains caused disease and death of the scale insect, among which L. lecanii displayed stronger virulence than F. incarnatum-equiseti to M. matsumurae. Furthermore, L. lecanii V3.4505 was most virulent and it caused the mortality of 61.33% and 100% to the second instar nymph and the adult females, respectively. The susceptibility of different instar to L. lecanii, V 3.4505, was varied. The adult females and the male 3rd-instar nymphs of M. matsumurae were susceptible to L. lecanii V3.4505; the adult females were more susceptible at LD50=1.96 than the 3rd-instar nymphs at LD50=5.67. The reason of the different susceptibility infected by the pathogens was discussed based on the morphology, body surface structure, cuticle thickness and wax secretions of M. matsumurae. The newly emerged male 3rd-instar nymphs and adult females are identified as a crucial period of the insect’s life cycle for M. matsumurae bio-control using pathogens. This is the first report on the infection of M. matsumurae by pathogens.4. Histopathological and glucose metabolic changes of Matsucoccus matsumurae after infected with Lecanicillium lecaniiThe infection process and histopathological changes of Matsucoccus matsumurae infected with Lecanicillium lecanii were studied using light microscopy, scanning and transmission electron microscopy. The results revealed that, L. lecanii invaded the scale insect rapidly and mainly by penetrating integument. After attached on the body surface, the conidia germinated soon.12h after inoculation, the hyphae were visible on the insect surface. The hyphae mainly distributed on the intersegmental folds, dorsal cicatrices, the base of the legs and vulva.24h after inoculation, it was observed that the hyphae had penetrated the integument and made the protein and chitin complex changed. After entered into the hemocoele, the hyphae invaded the hemocytes firstly and made cell membranes disrupted and the organelle disappeared. Then, the hyphae invaded the internal tissues. The attachment and infection of the hyphae to the muscle was observed and that resulted in the muscle anamorphosis and the muscle fiber loose. Many hyphae also invaded the fat bodies that caused the membrane of the fat cells disappearing and lipid droplets from different cells fused together. The ovarioles occupied most part of the hemocoele. It was observed that the hyphae attached on and invaded the follicular cells, and caused the follicular cells separated from the oocytes. The hyphae propagated in the hemocoele and exhausted the nutrition of the insect, then, they protruded from the insect integument and spread to the outside. It was also found that the fungus entered into the insect body via the vulva and infected the ovaries and the tissues nearby.The activity of trehalase and concentration of trehalose and glucose in hemolymph in M. matsumurae were assayed using biochemical method. The results showed that the trehalase activity increased firstly after the insect was infected with L. lecanii, and after 12h, the trehalase activity in the treated group was higher than that in control group. After 36h, the enzyme activity reached maximum of (0.480±0.020) U/mg, which is about 1.5 times to the control group. Then the trehalase activity decreased rapidly. Simultaneously, the concentration of trehalose decreased gradually. After 24h, it was lower than the control group, and after 96 h, it became only (0.623±0.047) mM. In contrast, the concentration of glucose increased rapidly and reached maximum of (0.704±0.029) mM 36h after inoculation, then it reduced gradually. These changes of the three factors indicated that the fungal infection induced the trehalase activity, which degraded the trehalose to glucose and a series of reaction between trehalase-trehalose-glucose. At last, the nutrition inside the insect body was exhausted by the pathogen, and the insect dead.These results provided evidence from both histopathology and sugar balance to reveal how M. matsumurae was infected and dead by L. lecanii.The results on the study of Ceroplastes japonicus are as follows:5. Invasion mechanism of the pathogens to the wax test of Ceroplastes japonicusIn order to investigate the invasion mechanism of fungal pathogens to the wax test of scale insects, strains of Lecanicillium lecanii (V3.4504 and V3.4505) and Fusarium incarnatum-equiseti (HEB01 and HEB02) were employed, and adult female of Ceroplastes japonicus with a large amount of wax secretion was used as the target.The results showed that L lecanii can penetrate through the wax test and invaded into the insect body successfully. However, the fungal growth, propagation and invasion on the wax test were much slower than on the insect body removed wax test. It indicated that the wax test had the function to inhibit the fungal invasion in a certain degree. The whole wax test, single wax substance and single honeydew, as the sole carbon source, were used to prepare three types of cultural media, respectively. It was found that all of the four strains germinated and developed into colonies on the three types of media. This phenomenon means that the fungi degrade the wax test and utilize the components of the wax test as their nutrition. Under the action of the fungi, the degradation rate of the whole wax test (17.23%-18.70%) was higher than that of the single wax substances (7.15%-12.78%). It indicated that the pathogens not only used the components of the wax substances, but also took in honeydew as their nutrition. By using GC/MS analysis, it was found that the pathogens mainly degraded the ester ethanol and short chain carboxylic ester from the wax test. By assaying the activity of the esterase, it was found that the esterase secreted by the fungi involved in the wax test degradation, and the esterase activity has notable positive correlation with the mortality of the scale insect, with a regression equation:y= 0.0127x-0.6605 (R2=0.9431). These results revealed the degradation mechanism of the pathogens to the wax test.6. Penetration of the pathogens to integument of Ceroplastes japonicus and role of extracellular enzymesIn order to study penetration of the pathogens to integument of Ceroplastes japonicus and role of extracellular enzymes secreted from the fungi, the WGA/Ovi-G labelling technique and biochemical assay were used. By using transmission electron microscopy, it was observed that the colloidal gold combined successfully to the chitin in the procuticle of the scale insect, so that the chitin showed its structure in a parallel sheet, and the chitin microfibrils in an evenly helical arrangement. The hisotopathological symptom was observed on the chitin with the structural anamorphosis and disruption of chitin sheet in the procuticle penetrated by the fungus. The gold particles labelled to chitin around the hyphae reduced, which indicated that the chitin was degraded by the extracellular enzymes secreted from the fungi.The activities of subtilisin-like protease, chitinase, p-N-acetyl glucosidase and lipase of the pathogens cultured on the cuticle substances were assayed by using microplate reader. The results showed that all of these four enzymes were involved in the cuticle degradation of the scale insect during fungal infection. Of the four enzymes, activity of lipase occurred earliest, and its maximum appeared in two days. The activity of subtilisin-like protease followed and its maximum appeared in three days. After that, the activities of chitinase and β-N-acetyl glucosidase emerged in five or four days. These results indicated the order of the action of the four enzymes during cuticle degradation:the pathogens firstly secreted lipase to degrade the lipid in the epicuticle, then they invade the procuticle and their protease was induced to degrade the protein, with the chitin matrix exposing, the chitinase degraded the chitin. Under the combined action of these enzymes the cuticle was degraded, which enhanced the fungal penetration through the insect integument. Reduction of the gold particles around the hyphae demonstrated degradation action of the two chitinase. The mortality of Ceroplastes japonicus correlated positively with the activity of NAG (R2= 0.9109).7. Infection of the pathogen to the hemolymph in CeroplastesJaponicusThe infection of L. lecanii to the hemocytes in hemocoele was studied using transmission electron microscopy. On the samples of early infection, it was observed that the pathogens were phagocytosed and assimilated by plasmocytes and granulocytes; while on the samples of later infection, the pathogen invaded the hemocytes and resulted in cell membranes break and organelle disappeared. Many myelinbodies were also found in the damaged hemocytes.The activities of phenoloxidase, superoxide dismutase, peroxidase, catalase in the hemolymph of the scale insects were assayed using microplate reader. The results indicated that the maximum value of the activity of the four enzymes emerged in three days after infection, while the activity of these enzymes decreased at the later stage. Meanwhile, the trehalase activity, and the content of the trehalose and glucose were measured. They also showed a trend of higher at the initial stage of infection and lower at the later stage, and the content changes of the trehalose and glucose correlated significantly with the trehalase activity. These results indicated that the fungal infection induced the immune response and affected the glucose metabolism of the scale insect, which is an important reason of the scale insect diseased. The present study is the first one to provide new evidence for the infection mechanism of the fungus to the hemolymph.