Pathogenicity of three entomopathogenic fungi to Matsucoccus matsumurae.

Matsucoccus matsumurae (Kuwana) (Hemiptera: Coccoidea: Matsucoccidae) is an invasive alien species and a destructive pest of two native Chinese pines, Pinus tabulaeformis Carr. and P. massoniana Lamb., throughout the eastern regions of China. The pathogenicity of three entomopathogenic fungi, Lecanicillium lecanii strain V3.4504 and V3.4505, Fusarium incarnatum-equiseti strain HEB01 and Lecanicillium fungicola strain 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 the L. lecanii strains V3.4504 and V3.4505 displayed stronger virulence than the F. incarnatum-equiseti strains HEB01 and L. fungicola strain HEB02 to M. matsumurae in the 2nd-instar nymphs and the adult females. Furthermore, L. lecanii V3.4505 was most virulent to M. matsumurae. 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 LT50 = 1.96 than the 3rd-instar nymphs at LT50 = 5.67. The body surface structure, cuticle thickness and wax secretions of M. matsumurae impacted the fungal infection. L. lecanii is a promising biocontrol agent, and newly emerged male 3rd-instar nymphs and adult females are a crucial period of the insect's life cycle for M. matsumurae biocontrol.

Ultra- and microstructure of the female reproductive system of Matsucoccus matsumurae.

The ultra- and microstructure of the female reproductive system of Matsucoccus matsumurae was studied using light microscopy, scanning and transmission electron microscopy. The results revealed that the female reproductive system of M. matsumurae is 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 contains 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 that becomes binucleate at the beginning of vitellogenesis. Accessory nuclei are observed in the peripheral ooplasm during vitellogenesis.

The wax glands and wax secretion of Matsucoccus matsumurae at different development stages.

In this paper, the wax secretions and wax glands of Matsucoccus matsumurae (Kuwana) at different instars were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The first and second instar nymphs were found to secrete wax filaments via the wax glands located in the atrium of the abdominal spiracles, which have a center open and a series of outer ring pores. The wax gland of the abdominal spiracle possesses a large central wax reservoir and several wax-secreting cells. Third-instar male nymphs secreted long and translucent wax filaments from monolocular, biolocular, trilocular and quadrilocular pores to form twine into cocoons. The adult male secreted long and straight wax filaments in bundles from a group of 18-19 wax-secreting tubular ducts on the abdominal segment VII. Each tube duct contained five or six wax pores. The adult female has dorsal cicatrices distributed in rows, many biolocular tubular ducts and multilocular disc pores with 8-12 loculi secreting wax filaments that form the egg sac, and a rare type wax pores with 10 loculi secreting 10 straight, hollow wax filaments. The ultrastructure and cytological characteristics of the wax glands include wax-secreting cells with a large nucleus, multiple mitochondria and several rough endoplasmic reticulum. The functions of the wax glands and wax secretions are discussed.

The effect of Beauveria brongniartii and its secondary metabolites on the detoxification enzymes of the pine caterpillar, Dendrolimus tabulaeformis.

The mortality of pine caterpillar, Dendrolimus tabulaeformis Tsai et Liu (Lepidoptera: Lasiocampidae), larvae treated with Beauveria brongniartii (Saccardo) Petch (Hypocreales: Clavicipitaceae) conidia and cell-free culture supernatants enriched for the secondary metabolites of the fungus was investigated. In addition, the effects of the treatments on the activities of two insect-related defense response proteins, glutathione S-transferase (GST) and esterase (EST), were measured over time. Bioassays were performed using a range of fungal spore (6 × 105 through 6 × 107 spores/mL) and supernatant extract concentrations (5.5-550 µg/mL). The results showed that the mortalities of D. tabulaeformis larvae were closely related to the concentration of the conidia and the metabolites of B. brongniartii. The differences among the treatments all reached a significant level. The activities of the two detoxifying enzymes, GST and EST, in the larvae increased simultaneously post-treatment. After infection with the conidial suspensions, the highest GST activity appeared at 3 days, and the activities of the caterpillars infected with 6 × 106 spores/mL and 6 × 107 spores/mL were significantly higher than in the control. Using α-naphthyl, the highest activity of EST also appeared at 3 days, and the differences for the three different concentrations were significant. A similar trend of change in the EST activity was observed using ß-naphthyl. After treatment with the secondary metabolite solution, the highest GST activity appeared at 6 hr, and significant differences were found both for the different durations (2, 4, 6, 12, 24, and 48 hr) and in the three concentration groups. When using α-naphthyl, the EST activity peak appeared at 24 hr, and the differences were significant among the durations of 2, 4, 6, 12, 24, and 48 hr. The effect of the concentration of the secondary metabolite solution notably induced the EST activity in the insects, and a similar result was obtained using ß-naphthyl. The data suggest that B. brongniartii produces secondary metabolites that disable the immune mechanisms of D. tabulaeformis, allowing the fungus to overcome and then kill its host. It was concluded that both the conidial suspensions and the metabolites of B. brongniartii were toxic to D. tabulaeformis larvae.

Cellular apoptosis of hemocytes from Dendrolimus tabulaeformis Tsai et Liu larvae induced with the secondary metabolites of Beauveria brongniartii (Sacc.) Petch.

To investigate the effect of the secondary metabolites of entomopathogenic fungus on the hemocyte immunity of host insect, the secondary metabolite complex (SMC) of Beauveriabrongniartii was used in three concentrations (5.5, 55, and 550 µg/mL), and the 4(th) instar larvae of the pine caterpillar Dendrolimustabulaeformis were employed as host insects. The larvae were inoculated with the SMC solutions by injection in bioassays. Apoptosis of the larval hemocytes was observed using fluorescence microscopy (FM), transmission electron microscopy (TEM), and flow cytometry (FCM). The FM results showed that in the treated groups, larval hemocytes exhibited symptoms of early apoptosis at 6 h post-treatment by radiating a non-uniform kelly fluorescence and exhibited symptoms of late apoptosis at 12 h post-treatment by radiating a non-uniform orange fluorescence. Under TEM, the following ultra-structural changes associated with apoptosis of the larval hemocytes were observed in the treated groups: the nuclei were hypertrophied, slight folds were on the nuclear envelope, the chromatin became concentrated, the mitochondrial cristae disappeared or were disorderly, most cells developed blebs, and fibrillar aggregation appeared and accumulated in the cytoplasm. Apoptosis of the larval hemocytes was detected by FCM at 6 h post-treatment; the percentage of early apoptotic cells in the SMC 5.5, 55, and 550 µg/mL treatment groups were 11.93%, 13.10%, and 18.42%, respectively. Late apoptosis first occurred at 12 h post-treatment; the highest rate of apoptosis was 36.54 ± 4.37% at 24 h post-treatment in the SMC 55 µg/mL treatment group. In general, the cellular apoptosis rate was positively correlated with the SMC concentration and the time post-treatment. These results indicate that secondary metabolites of B. brongniartii are able to attack the hemocytes of D. tabulaeformis larvae and induce cellular apoptosis, thereby providing new evidence that secondary metabolites of mycopathogens can act on host immune systems.

[Use of entomopathogenic fungi to degrade wax secreted by Ceroplastes japonicus].

OBJECTIVE: We used entomopathogenic fungi to degrade insect wax. METHODS: We used four fungal strains, Lecanicilliurn lecanii V3. 4504, V3. 4505, Beauveria bassiana FDB01, and Metarhizium anisopliae TSL06. Wax coverings of female adults of Ceroplastes japonicus Green (Insecta: Hemiptera: Coccoidea) were used as the sole carbon source in the mineral medium. RESULTS: All of the 4 strains could grow, reproduce, produce enzymes, and degrade wax. During a 7-day culture, the highest lipase activities of the 4 strains, V3. 4504, V3. 4505, FDB01, and TSL06 were 0.128 +/- 0.017, 0.056 +/- 0.002, 0.124 +/- 0.011, and 0.149 +/- 0.005 U/mL, respectively. The dehydrogenases activities of the 4 strains were 0.075 +/- 0.003, 0.074 +/- 0.003, 0.061 +/- 0.04, and 0. 066 +/- 0. 002 U/mL respectively. The degradation rates of wax by the 4 strains were 18.20 +/- 0.019, 11.00 +/- 0.011, 15.4 +/- 0.017, and 23.10 +/- 0.031%, respectively. CONCLUSION: The 4 strains could depredate wax of C. japonicus.

Morphological and ultrastructural characterization of Carposina sasakii larvae (Lepidoptera: Carposinidae) infected by Beauveria bassiana (Ascomycota: Hypocreales: Clavicipitaceae).

The aim of this study was to better understand the pathogenesis of the entomopathogenic fungus Beauveria bassiana (Balsamo) strain TST05 observed on the peach fruit moth (Carposina sasakii (Matsumura)), an important orchard pest. The morphological and ultrastructural characterization of the mature larvae of C. sasakii infected by B. bassiana was investigated by using light, scanning and transmission electron microscopy. The results of the study show that B. bassiana TST05 infected the host larvae mainly by penetrating the integument. The conidia of the fungus adhere easily to the area around the mouthparts and to the basal area around the acanthae on the thorax and abdomen. Observations of the host's defensive response to the fungal attack indicated that dark spots appeared on the cuticle and that melanization appeared in the hemocoel. After overcoming the host's defense system, the pathogen grew and reproduced primarily in the hemocoel. The infection spread sequentially to the internal tissues, e.g., fat body, muscle, Malpighian tubules, gut and even the silk gland. Ultimately, the larval internal organs and tissues were damaged very extensively. Finally, the fungus emerged through the cuticle of the dead insect and released conidiophores that could act as new pathogens to infect other larvae.

The structure of integument and wax glands of Phenacoccus fraxinus (Hemiptera: Coccoidea: Pseudococcidae).

Using scanning electron microscopy and optical microscopy, we studied the structure of the integument and wax glands of the mealybug, Phenacoccus fraxinus Tang (Hemiptera: Coccoidea: Pseudococcidae). We observed the ultrastructure of four wax pores including trilocular, quinquelocular, and multilocular pores as well as tubular ducts, recording characteristics of their structure, size and distribution. We found that that the integument of the mealybug consists of three main layers-the procuticle, epidermis and basement membrane-and four sub-layers of the procuticle-the epicuticle, exocuticle, endocuticle and formation zone. The wax-secreting gland cells were closely arranged in epidermis. All of them were complex and composed of one central cell and two or more lateral cells. These complex cells possess a large common reservoir for collection and storage. Synthesized by the glandular cells, the wax is excreted outside integument through canals.

Morphological and ultrastructural characterization of the alimentary canal in Japanese wax scale (Ceroplastes japonicus Green).

The morphology and ultrastructure of the alimentary canal in the adult female of the Japanese wax scale, Ceroplastes japonicus Green (Hemiptera: Coccoidea: Coccidae), was investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The results showed that the foregut was subdivided into a sclerotized pharynx and an oesophagus. A pair of salivary glands attached in the middle of the foregut. The loop-shaped midgut was narrow and longer than the foregut and its inner wall lined with a thick layer of epithelia. The hindgut was divided into a narrower ileum and a broader rectum, with the well-developed filter chamber enclosed in the anterior rectum. Malpighian tubules consisted of two brownish-yellow moniliform tubules with pores, approximately 1 µm in diameter, scattered on the outer surface and many spherical crystals inside the tubules.

Ultrastructural and cytochemical characterization of brown soft scale Coccus hesperidum (Hemiptera: Coccidae) infected by the Lecanicillium lecanii (Ascomycota: Hypocreales).

The ultrastructural and cytochemical characterization of the brown soft scale, Coccus hesperidum L. (Hemiptera: Coccidae) infected by the hyphomycete Lecanicillium lecanii (Zimmermann) Gams & Zare, belonging to the phylum Ascomycota and order Hypocreales, was investigated by light, scanning and transmission electron microscopy. Gold cytochemistry was used to label chitin in the cuticle of the scale insect. The results revealed that the pathogenic fungus, L. lecanii generally infected by penetrating the integument, especially at anus, vulva, spiracles, stigmatic furrow, body margin, and the areas of cuticle with grooves, fissures and rugoses areas. The conidia became attached to the host body surface and germinated into hyphae that established colonies by branching repeatedly. Hyphae penetrated the integument by means of their penetration pegs using mechanical force and extracellular enzymes. During integument penetration, the hyphae extended vertically or parallel along the cuticle. Labeling with the WGA/Ovo-G complex showed disruption of the parallel sheets of chitin and a decrease in the density of the gold particles surrounding the penetrated hyphae. Hyphal invasion also separated the cuticle and epidermis from each other. Once in the haemocoele, blastospores of the fungus infected the haemocytes and internal organs. After some time, the nutritive value of the haemocoele decreased and the insect's internal organs disappeared. The hyphae then produced conidiophores and released them through the cuticle of the scale insect cadaver.

[Effect of multi-generation culture on virulence of Lecanicilliurn lecanii in different media].

OBJECTIVE: The strain No. V3.4504 of Lecanicilliurn lecanii (Zimmermann), an entomopathogenic fungus, was studied on the effect of successive multi-generation culture in seven different media on its colony growth characteristics, extracellular enzyme activities and the virulence against scale insects. METHODS: The strain No. V3.4504 of L. lecanii was original isolated from a natural infected scale insect. The two species of scale insects used were Rhodococcus sariuoni Borchsenius and Ceroplastes japonicus Green. Seven media were used and fungus colony characteristics, growth rate and sporulation, extracellular protease and chitinase activity, and infective effect against the two species of scale insects were conducted. RESULTS: The fungus cultured on PDA medium for successive nine generations showed the most fast in colony growth, the minimal in sporulation, straight decline of extracellular protease and chitinase activity with generation increasing, and the minimal mortality of the scale insects. There was no significant effect to promote virulence of the fungus by increasing peptone into medium. On the media D, E and F, that with the body materials of the two scale insects, although the fungus appeared lower in the colony growth rate, its sporulation was higher upward 8.83 x 10(6) - 9.13 x 10(6) spores/cm2, extracellular protease and chitinase activities averagely reached 2.16 - 2.13 U/g and 1.01 - 1.03 U/g respectively, and the mortalities of the two scale insects were 55% - 58% and 39% - 42% respectively. Cultured three generations in vitro of the two scale insects, the fungus exhibited the highest activities in its protease and chitinase that were 3.08 - 2.92 U/g and 1.45 - 1.42 U/g respectively and the best infection effect against the two scale insects with mortalities of 71.30% and 58.89% respectively. A linear correlation was found between extracellular protease and chitinase activities of the fungus and the mortalities of the scale insects. CONCLUSION: Cultured on PDA medium successive multiple generations made retrogradation of the strain No. V3.4504 of of L. lecanii. It was significant effect on keeping the vigor and higher virulence of the fungus adding the body materials of the scale insects into the medium. The vitro by using live scale insects as medium materials was the best way for the rejuvenation of the entomopathogenic fungus and promoting its virulence.

Effect of volatile emissions, especially alpha-pinene, from persimmon trees infested by Japanese wax scales or treated with methyl jasmonate on recruitment of ladybeetle predators.

Persimmon, Diospyros kaki L., is an important fruit tree in northern China. Japanese wax scale, Ceroplastes japonicus Green (Hemiptera: Coccoidea: Coccidae), is the most destructive pest in persimmon orchards and is difficult to control using chemical pesticides. This paper studied the variety of volatile emissions from persimmon trees attacked by wax scale or induced by applications of methyl jasmonate (MeJA), an exogenous signaling material. We also studied the role of volatiles in recruiting the ladybeetle, Chilocorus kuwanae Silvestri (Coleoptera: Coccinellidae), a primary predator of wax scale. The results showed that the ladybeetles displayed strong taxis responses to the odor sources from trees treated with MeJA and wax scale-damaged trees. Within 24 h after the trees were treated with MeJA, the number of ladybeetles responding to the treated trees fluctuated based on the amount of volatiles released over a diel period. Chemical analysis of the volatiles showed that terpenoid compounds, especially alpha-pinene, increased from both the wax scale-damaged and MeJA-treated trees and that this was the reason for the ladybeetle attraction. Therefore, alpha-pinene was used as a single signal source to examine the taxis response of C. kuwanae. The results suggest that alpha-pinene plays a significant role in attracting the ladybeetles. It is therefore concluded that MeJA application might be used to stimulate the defense function of persimmon trees by inducing the release of terpenoids, especially alpha-pinene, and thereby recruiting more ladybeetles to control the scale insects.

Histopathological changes of Ceroplastes japonicus infected by Lecanicillium lecanii.

The infection process and pathological changes of Japanese wax scale, Ceroplastes japonicus Green, by the hyphomycete Lecanicillium lecanii (Zimmermann) Gams & Zare were investigated by light, scanning and transmission electron microscopy. The results showed that L. lecanii generally infected the wax scale by penetrating the integument. The anal area, the body margin, around the base of mouthparts and legs, over the stigmatic furrow and the area around the vulva were susceptible places, while the wax test had an inhibitory effect on L. lecanii. Within 24h after inoculation, conidia became attached to the cuticle, and within 48h, hyphae adhered to the integument of the scale and their tips differentiated into specialized infection pegs. Penetration of the cuticle occurred within 72h of inoculation; the fungus caused the insect cuticle to rupture and hyphae entered the insect body through these openings. Within 72h after inoculation, L. lecanii entered the hemocoele of the scale and formed blastospores. After 96h, blastospores were dispersed throughout the hemolymph and completely disrupted the hemocytes, resulting in damage of the cell nucleus and agglutination of chromatin. Concomitant to colonization of the hemolymph, the internal organs and tissues, e.g., tracheae, malpighian tubules and muscle fibers, were also infected. As the infection progressed, the wax test and body changed color from white and red, respectively, to yellowish. After 144h, the internal tissue structure was totally compromised and the insects died. After this time, new conidiophores bearing conidia were produced on the surface of the cadavers.

[Isolation and identification of toxins inhibiting Dentrolimus tabulaeformis from an antagonistic strain of Beauveria].

OBJECTIVE: We used submerged fermentation to cultivate a strain of the entomopathogenic fungus Beauveria tenella isolated from the infected larvae of Dentrolimus tabulaeformis in Pinus tabulaeformis forest in Chengde of Hebei Province in China. METHODS: We used ethyl acetate to extract antagonistic components from the fermentation broth and used silica gel column chromatography and GC/MS to separate and identify the components. RESULTS: Six compounds were obtained by silica gel column chromatography. The sixth compound had higher activity to kill the larvae of Dentrolimus tabulaeformis with a corrected mortality rate of 80%. Seventeen compounds were separated and identified by GC/MS in the 6th group, of which 3compounds were more than 10%, 2-Piperidinone (14.02%), 2-coumaranone (47.10%), and Pyrrolo[1,2-a]Pyrazine-1,4-dione, hexahydro (21.05%). CONCLUSION: 2-Piperidinone and 2-coumaranone had insecticidal activity (corrected mortality rate reached 83.32% and 91.61% respectively) and were the most important toxic substances to control pests.