台湾虫草的被毛孢无性型新种及其对黄粉虫的侵染研究

报道采自安徽省黄山风景区的台湾虫草Cordycepsformosana,多批次收集弹射的子囊孢子分离得到其无性型,经鉴定为一被毛孢新种,命名为黄山被毛孢Hirsutellahuangshanensis。黄山被毛孢在米饭培养和自制的虫草培养基上均能长出子实体,其菌落及子实体颜色与天然虫草较为相似,但未见成熟的子囊壳。应用黄山被毛孢RCEF0868菌株对饲喂昆虫黄粉虫进行感染试验,结果表明:黄粉虫在菌落接触感染、刚蜕皮幼虫的穿刺接种和孢子浓度1.0×107/ml浸蘸处理下,获得了相对较高的侵染率,分别达到30%、25%和25%,刚蜕皮幼虫一般比深色幼虫组其侵染率较高,1×105/ml孢子浓度浸蘸试虫不论是刚蜕皮或老熟幼虫均不能感染。10d后被感染的黄粉虫形成僵虫,继续保湿培养20d后,体表节间膜处形成白色菌膜,粘片镜检表明感染真菌即为供试菌株。40d后,可见子实体原基及孢梗束,尚未见成熟的子囊壳。     

Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system

Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and β-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both silkworm BmN cell line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities of EG I that the silkworm provides maybe greatly facilitate the future research and the potential application in industries.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.     

Costs of cannibalism in the presence of an iridovirus pathogen of Spodoptera frugiperda

Abstract.  1. The costs of cannibalism were examined in larvae of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in the presence of conspecifics infected by a lethal invertebrate iridescent virus (IIV). The hypothesis of a positive correlation between insect density and the likelihood of disease transmission by cannibalism was examined in laboratory microcosms and a field experiment.
2. Transmission was negligible following peroral infection of early instars with purified virus suspensions or following coprophagy of virus-contaminated faeces excreted by infected insects. In contrast, 92% of the insects that predated infected conspecifics acquired the infection and died prior to adult emergence in the laboratory. Diseased larvae were more likely to be victims of cannibalism than healthy larvae.
3. The prevalence of cannibalism was density dependent in laboratory microcosms with a low density (10 healthy insects + one infected insect) or high density (30 healthy insects + one infected insect) of insects, and field experiments performed on maize plants infested with one or four healthy insects + one infected insect.
4. Cannibalism in the presence of virus-infected conspecifics was highly costly to S. frugiperda ; in all cases, insect survival was reduced by between ≈ 50% (laboratory) and ≈ 30% (field) in the presence of the pathogen. Contrary to expectations, the prevalence of disease was not sensitive to density because cannibalism resulted in self-thinning. As infected individuals are consumed and disappear from the population, the prevalence of disease will be determined by the timescale over which transmission can be achieved, and the rate at which individuals that have acquired an infection become themselves infectious to conspecific predators.     

Nutrient reserves in starving black carpet beetle larvae infected with the eugregarine Pyxinia frenzeli

Starvation-mediated alterations of nutrient reserves (lipid, protein, glycogen) of larvae of the black carpet beetle, Attagenus megatoma (=Attagenus piceus), were studied. Quantitative comparisons of these materials between uninfected larvae and those infected with the eugregarine Pyxinia frenzeli were performed in an effort to characterize pathologic conditions elicited by the gregarine. Fresh weight losses were similar in both types of insects; however, infected larvae had a consistently higher moisture content. No differences were detected in the rate of utilization of stored protein, glycogen, or lipid. These data provide evidence that P. frenzeli is a commensal or mutualist, rather than a pathogen.     

Common Leafspot of Alfalfa: Ascospore Germination and Disease Development in Relation to Moisture and Temperature

In a moist chamber Pseudopeziza medicaginis ascospores infected alfalfa (Medi sativa L.) moderately to abundantly within 6–10 h at 10–20 °C and within a longer time-span outside this temperature range. Approximate limits of the range were 2.5 and 28 °C; no infection took place at 30 °C. At 14°C ascospores infected alfalfa abundantly at 98 %relative humidity (RH) and above, moderately at 97%, sparsely at 95 and 96%, but not at 94% and below. Ascospores were hydrophilic, germinating best at or near 100%, RH but did not germinate at or below 93 % RH. After infection was established, tiny leafspots became visible within 6–7 days at constant temperatures of 15–25°, 10 days of 10°C, 13 days of 5 °C, and 25 days of 2.5 °C. They failed to develop into normal size spots within 4 weeks at constant temperatures near 30 °C, or near 10 °C and lower. Temporary exposure of incipiently diseased plants 1–6 days to 30–38 °C adversely affected subsequent leafspot development at 20–24°C. Inhibition depended on temperature and on the extent of post-infection disease development.     

Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: Winter profiles of a natural population

Larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) utilize a freeze-avoidance strategy for winter survival. Cold-hardiness adaptations of an outdoor population of the species were profiled over the 1984–1985 winter. Over the autumn months supercooling points of the larvae dropped from −13.9±2.3°C to −37.8±2.8°C (the lowest winter temperature recorded was −26°C), water content of the larvae decreased from 57.2±1.2 to 24.8±1.6% of fresh weight, and glycerol content of the larvae rose to an average of 2030 μmol/g wet weight or 18.7% of fresh weight. All parameters stabilized over the mid-winter months. Glycerol production was largely accounted for by the loss of stored glycogen while lipid and protein reserves remained nearly constant over the winter months. Supercooling-point depression and glycerol systhesis both appeared to be initiated after the first overnight exposures to subzero temperatures. Highest rates of glycerol production, about 60 μmol g⁻¹ d⁻¹, were achieved with mean daily temperatures of about 0°C and subzero nights. Glycerol content was rapidly cleared in the spring but only 20% of the resulting carbon was restored as glycogen.     

Effects of acclimation and diapause on the cold tolerance of Trogoderma granarium

Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), is a pest of stored grain in Africa, Asia, and Europe. It is a quarantine insect for much of the rest of the world. Control of T. granarium can be achieved with methyl bromide, but this fumigant is an ozone‐depleting substance and is being phased out worldwide. Thus, there is an urgent need to find new methods of control, including the use of low temperatures. Here, we assess the effects of diapause and cold acclimation on the cold tolerance of T. granarium. The percentage of larvae in diapause increased with larval density, reaching 57.3% when reared at a density of 73 larvae g^?1 diet. The cold tolerance of T. granarium was assessed by the supercooling points (SCPs) of various life stages. The SCP of non‐acclimated insects ranged from ?26.2 ± 0.2 °C (mean ± SEM) for eggs to ?14.4 ± 0.4 °C for larvae. The lowest SCP for larvae, ?24.3 ± 0.3 °C, was obtained for diapausing‐acclimated larvae. Based on mean LT₅₀ values, the most cold‐tolerant stage at ?10 °C was the diapausing‐acclimated larvae (87 days) followed by non‐diapausing‐acclimated larvae (51 days), diapausing non‐acclimated larvae (19 days), adults (4 days), non‐diapausing non‐acclimated larvae (2 days), pupae (0.4 days), and eggs (0.2 days). The estimated times to obtain 99.9968% mortality (Probit 9) for diapausing‐acclimated larvae are 999, 442, 347, 84, and 15 days at 0, ?5, ?10, ?15, and ?20 °C, respectively. Probit 9 is an estimated value used by quarantine experts to estimate conditions that are required to kill all insects. In light of the long exposure time needed to control T. granarium even at ?20 °C, cooling to below ?27 °C (i.e., below the SCP of eggs) will quickly kill all life stages and may be the best way to control this insect with low temperatures.     

Metabolic reserves associated with pupal diapause in the flesh fly, Sarcophaga crassipalpis

Larvae of Sarcophaga crassipalpis destined for pupal diapause (light:dark 12:12, 20°C) contain nearly twice as much lipid and twice the haemolymph protein concentration as larvae that will not enter diapause (light:dark 15:9, 20°C). This conspicuous difference in metabolic reserves provides the earliest indication of the developmental fate of the larva. Lipid reserves are utilized rapidly during the first half of diapause and then remain stable until adult eclosion. In contrast, residual dry weight changes very little early in diapause but drops sharply late in diapause, thus implying a transition from lipid utilization to protein or carbohydrate utilization in mid-diapause. We suggest that this metabolic transition marks the end of the “fixed latency period”: pupae readily respond to environmental or hormonal stimulation after this point. Diapause-destined larvae did not accumulate more glycogen than nondiapause-destined larvae, but an 80% decrease in glycogen at the onset of diapause and its elevation at the end of diapause suggests the utilization of glycerol or related compounds as cryoprotectants during diapause. Profiles of water content are very similar in short-day and long-day flies, thus suggesting that dehydration is not a mechanism exploited by the flesh fly to achieve cold hardiness. Adult flies that have experienced pupal diapause emerge from the puparium with lipid, glycogen, and water content nearly identical to flies that have not experienced diapause, but the residual dry weight is much lower. The severe depletion of protein may account for the reduced fecundity of flies that have experienced diapause.     

Histopathological changes in livers of mice infected with tetrathyridia of Mesocestoides corti and exposed to different environmental temperatures

Novak M. 1982. Histopathological changes in livers of mice infected with tetrathyridia of Mesocestoides corti and exposed to different environmental temperatures. International Journal for Parasitology12: 41–45. Observations on the histopathology of the liver of mice infected with Mesocestoides corti and kept for 20 days p.i. (post-infection) at low (5 ± 1°C), room (21 ± 1°C) or high (35 ± 1°C) temperature revealed that the degree of liver pathology was directly proportional to the intensity of liver infection, which in turn was the result of the temperature effect. The most severe pathological changes occured in the heavily infected organs of mice kept at low temperature, followed by less prominent changes in moderately infected livers of mice kept at room temperature and the smallest changes in lightly infected livers of mice kept at high temperature. The pathological changes in infected and uninfected livers of hosts exposed to different environmental temperatures are described and compared.     

Can plants mediate the humoral immunity and biochemical metabolism of entomopathogen‐infected caterpillars?

Plant‐insect herbivore‐entomopathogen interactions are one of the hot topics in biological control and humoral immunity, and biochemical metabolism are important responses of herbivores to pathogen infection. Entomopathogens are key biocontrol agents of caterpillars, but how plants affect the responses of caterpillars to these organisms is not well understood. We studied hormonal immunity (lysozyme and phenoloxidase activities) and biochemical metabolism (total protein and lipid contents) of Beauveria bassiana‐infected beet armyworm (Spodoptera exigua) larvae that feed on five different host plants (soya bean, Chinese cabbage, edible amaranth, water convolvulus and pepper). Results indicated that plant species differentially affected lysozyme and phenoloxidase activity and lipid content, but had no effect on protein content of pathogen‐infected caterpillars. Both lysozyme and phenoloxidase activities were generally higher in entomopathogen‐infected larvae that feed on edible amaranth or water convolvulus compared with the other three plants from days 1 to 5 after treatment. Plant species did not affect in regular changes during the 5 days in the lipid content of infected or non‐infected caterpillars. Our study reveals that plants fail to affect the biochemical metabolism but plants can mediate the humoral immunity of caterpillars to defend against pathogens. This study provides insight into plant‐mediated effects on the response of herbivores to pathogens.     

Food consumption by cabbage loopers infected with nuclear polyhedrosis virus

The effects of age, temperature, and dose on artificial medium consumption by healthy and nuclear polyhedrosis virus-infected cabbage looper larvae were measured using gravimetric methods. Instar in which lethal infection occurred was more closely related to subsequent food consumption than was larval age in days. Larval cabbage loopers, lethally infected in either the first or second instar, consumed 2% or less of their potential consumption. Larvae infected in the third instar consumed ca. 5% of their subsequent potential. In the fourth instar, this amount increased to ca. 10%. If infection occurred in the fifth instar, no significant amount of feeding was prevented. Increasing the virus dosage significantly decreased consumption and length of feeding period over the range of dosages tested. The relationship between consumption patterns of diseased and healthy insects remained constant over a 20–35°C temperature range.     

Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

Susceptibility of Phyllophaga polyphylla and Anomala cincta larvae to Beauveria bassiana and Metarhizium anisopliae isolates, and the interaction with soil properties

The white grub species Phyllophaga polyphylla and Anomala cincta (Coleoptera: Melolonthidae) are economically important species that affect many crops in Mexico. A series of experiments to study the pathogenic interaction between isolates of Beauveria bassiana and Metarhizium anisopliae and these two insect species were undertaken. First, the susceptibility of third instar P. polyphylla larvae to each of seven isolates representing both species of fungus was evaluated by dipping the insects in 1?×?10⁸ conidia?ml^?1 suspensions. A second study examined the differences in the susceptibility of P. polyphylla and A. cincta larvae to two selected isolates for each of the fungal species. Finally, the susceptibility of A. cincta larvae to one M. anisopliae isolate when incubated in soil collected from four different sites was assessed. No significant differences in proportion of infection of P. polyphylla larvae were observed amongst the fungal isolates tested and mortality due to fungal infection was never greater than 20% after 36?days incubation. Anomala cincta larvae were more susceptible than P. polyphylla larvae, with greater than 90% infection when inoculated with isolates of M. anisopliae whereas mortalities of only 20% where achieved against P. polyphylla larvae. The soil type in which A. cincta were incubated following inoculation with M. anisopliae affected their susceptibility to infection. The results demonstrated that there is a complex interaction amongst entomopathogenic fungi, white grub larvae and soil properties, and points to the need of further investigation of this system in order to optimize the efficacy of entomopathogenic fungi against these insect species.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

The susceptibility ofTribolium castaneum [Col.: Tenchrionidae] toFarinocystis tribolii [Protozoa: Schizogregarinida]

The LT₅₀ ofFarinocystis tribolii Weiser to larvae ofTribolium castaneum (Herbst) increased with the age of the insect indicating that older larvae were relatively more tolerant to the infection though there was 100 % mortality ultimately. The adults were less susceptible than larvae and between sexes, females were more susceptible than males. The number of spores produced increased with the stage of the larvae, but there was no variation in the size of spores in the different instars. The LC₅₀ on 20th and 40th day of inoculation were 1.4×10⁷ and 2.1×10⁶ respectively. Mortality-time due toF. tribolii was shorter at 35 °C than at 25 °C. Sporulation occurred earlier at 35 °C than at 25 °C.     

Acid and alkaline phosphatase activity in the fat body and midgut of the beet armyworm,Spodoptera exigua (Lepidoptera: Noctuidae), infected with nuclear polyhedrosis virus

Changes in the activity of acid and alkaline phosphatase in Spodoptera exigua larvae infected with nuclear polyhedrosis virus have been investigated. Three days after per os infection, the activity of acid phosphatase in the fat body and midgut of infected larvae was significantly higher than that in normal larvae. Alkaline phosphatase activity did not show such significant changes. There were differences in the phosphatase patterns depending on whether their activities were expressed as enzyme units per milligram of fresh organ weight or per milligram of homogenate protein. The literature relevant to the subject allows us to conclude that the increase in phosphatase activities in S. exigua larvae is not specifically associated with virus infection itself, but, rather, is a reaction of the insect organism to the diminishing supply of energy sources.     

Iron Levels Change in Larval Heliothis virescens Tissues Following Baculovirus Infection

Inductively coupled plasma mass spectrometry and (59)Fe radiotracers were used to investigate changes in levels of Fe in the tissues of 4th instar Heliothis virescens larvae following infection with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or with Autographa californica multiple nucleopolyhedrovirus. Baculovirus infection led to significant changes in hemolymph Fe levels late in infection. (24)Na radiotracer ingested by 4th instar larvae was rapidly cleared to nearly undetectable levels 6 h post-ingestion. In contrast, (59)Fe radiotracer fed to 4th instar larvae declined within the first few hours of ingestion and then remained constant at approximately 60% of the initial tracer activity. While Fe radiotracer levels among larval tissues changed, whole insect tracer levels did not decline from 6 to 60 h post-ingestion. Tissues from HzSNPV larvae had higher radiotracer levels in the hemolymph and midgut 36 and 60 h post-infection. The protein-bound/free ratio of (59)Fe was significantly higher in baculovirus infected hemolymph than in uninfected hemolymph at 60 h post-infection, indicating that Fe released from damaged cells is protein-bound. In both studies, hemolymph Fe levels were higher in HzSNPV infected larvae. This first study of tissue Fe levels during viral infection of an insect clearly demonstrates that Fe homeostasis is substantially disrupted.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.