Interactions between environmental variables determine immunity in the Indian meal moth Plodia interpunctella

1. Animals raised in good environmental conditions are expected to have more resources to invest in immunity than those raised in poor conditions. Variation in immune activity and parasite resistance in response to changes in environmental temperature, population density and food quality have been shown in many invertebrate species. 2. Almost all studies to date have examined the effects of individual variables in isolation. The aim of this study was to address whether environmental factors interact to produce synergistic effects on phenoloxidase (PO) activity and haemocyte count, both indicators of immune system activity. Temperature, food quality and density were varied in a fully factorial design for a total of eight treatment combinations. 3. Strong interactions between the three environmental variables led to the magnitude and in some cases the direction of the effect of most variables changing as the other environmental factors were altered. Overall, food quality had the most important and consistent influence, larvae raised on a good-quality diet having substantially higher PO activity in every case and substantially higher haemocyte counts in all treatments except unheated/low density. 4. When food quality was good, the larvae showed 'density-dependent prophylaxis': raising their investment in immunity when population density is high. When food quality was poor and the temperature low, however, those larvae raised at high densities invested less in immunity. 5. Increased temperature is often thought to lead to increased immune reactivity in ectotherms, but we found that the effect of temperature was strongly dependent on the values of other environmental variables. PO activity increased with temperature when larvae were raised on good food or when density was high, but when food was poor and density low, a higher temperature led to reduced PO activity. A higher temperature led to higher haemocyte counts when density was high and food quality was poor, but in all other cases, the effect of increased temperature was either close to zero or somewhat negative. 6. Although PO activity and haemocyte count were weakly correlated across the whole data set, there were a number of treatments where the two measures responded in different ways to environmental change. Overall, effect sizes for PO activity were substantially higher than those for haemocyte count, indicating that the different components of the immune system vary in their sensitivity to environmental change. 7. Predictions of the effect of environmental or population change on immunity and disease dynamics based on laboratory experiments that only investigate the effects of single variable are likely to be inaccurate or even entirely wrong.     

Toxicity of parasporal crystals of Bacillus thuringiensis to the Indian meal moth, Plodia interpunctella.

Toxicity of Bacillus thuringiensis parasporal crystals to the Indian meal moth, Plodia interpunctella, is described. The numbers of insects killed were in relation to crystal dry weight. Mortality was determined by comparing adult emergence in diets treated with crystals to emergence in untreated diets. There was only a 30% survival at an application of 0.414 microgram/cm2, and the mean 50% lethal concentration value was found to be 0.299 microgram/cm2. The use of emergence data has provided a reliable and reproducible bioassay for comparing relative toxicities of crystals, spores, and other cellular components to this economically important insect.     

Toxicity of parasporal crystals of Bacillus thuringiensis to the Indian meal moth, Plodia interpunctella.

Toxicity of Bacillus thuringiensis parasporal crystals to the Indian meal moth, Plodia interpunctella, is described. The numbers of insects killed were in relation to crystal dry weight. Mortality was determined by comparing adult emergence in diets treated with crystals to emergence in untreated diets. There was only a 30% survival at an application of 0.414 microgram/cm2, and the mean 50% lethal concentration value was found to be 0.299 microgram/cm2. The use of emergence data has provided a reliable and reproducible bioassay for comparing relative toxicities of crystals, spores, and other cellular components to this economically important insect.     

Cannibalism and the stage-dependent transmission of a viral pathogen of the Indian meal moth, Plodia interpunctella

1. The transmission of an insect pathogen by cannibalism was studied by a series of choice and no-choice experiments.
2. Infection of Plodia interpunctella larvae with a granulosis virus occurred through cannibalism of infected larvae.
3. Depending on the larval instars of the cannibal and the victim relative to each other, preferential cannibalism of both infected and healthy larvae was observed.
4. Larvae cannibalise healthy, less vulnerable larvae preferentially, but it is argued that there is no evidence that they are avoiding infection.
5. The victim cannibalised can be explained as a balance between the reduced risk of injury and the ease of cannibalism of moribund infected individuals on the one hand and the greater food resource value of healthy individuals on the other.
6. The implications for the insect population dynamics of the transmission of the pathogen via cannibalism and the effective removal of infective particles through cannibalism by immune stages are discussed.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.     

Resource limitation and the lethal and sublethal effects of a viral pathogen in the Indian meal moth, Plodia interpunctella

Abstract.

• 1 The effects of resource limitation and the lethal and sublethal effects of a granulosis virus on a lepidopteran host, the Indian meal moth, Plodia interpunctella, were examined.
• 2 The food quality was manipulated by the addition of an inert bulking agent (methyl cellulose) which caused the size, development rate and fecundity of the moths to be reduced.
• 3 The resource quality had no effect on the mortality due to the virus. In contrast, sublethal effects of the virus on pupal weight were more apparent under conditions of resource limitation.
• 4 Considerable variation between the sublethal effects after challenge with different doses of the virus was found. The balance between deleterious sublethal effects of the virus and the selection of more robust individuals by the bioassays is proposed as a mechanism to explain this variation.
• 5 Implications for the dynamics of insect hosts and their pathogens are discussed.

     

Effects of resource quality on the population dynamics of the Indian meal moth Plodia interpunctella and its granulovirus

Although the Plodia interpunctella-granulovirus system is one of the most studied models for insect-pathogen interactions, there are relatively few precise data on the dynamics of the virus in coexisting populations of these two organisms. Previous work has suggested that resource quality, in terms of the diet supplied to P. interpunctella, has a strong effect on the population dynamics of host and pathogen. Here we investigate the impact of resource-dependent host patterns of abundance on pathogen dynamics and prevalence. In the laboratory, three populations of P. interpunctella feeding on a good quality food and infected with a granulovirus were compared with three populations also infected with a granulovirus but feeding on poor quality food. Populations feeding on good quality food produced larger adult moths, and had greater numbers of adult moths, healthy larvae, and virus-infected larvae. A higher proportion of larvae in these good quality populations were infected with virus, and adult moths exhibited cyclic fluctuations in abundance, unlike those on poor quality food. This cyclic behaviour was shown to be associated with cycles in the age structure of the larval population. Previous theoretical work suggests that these cycles may result from asymmetric competition between young and old larvae. Cyclic fluctuations in the proportion of infected larvae, that occurred on good, but not on poor quality food, were also shown to be related to cycles in the age structure of the larval population.     

Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-component pheromone blend

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antennae upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e., (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9,12-tetradecadienal (Z9,E12-14:Ald) and (Z,E)-9,12-tetradecadienol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-11-hexadecenyl acetate (Z11-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests there were no significant differences in response of male P. interpunctella to the bait containing all four EAD-active compounds and the responses to female gland extacts. A behavioural assay of different two-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major pheromone component Z9,E12-14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the compounds from the complete four-compound blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of P. interpunctella consists of at least four components, i.e., Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH and Z9-14:OAc.In a field trapping test performed in a storage facility, the four-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc alone. In contrast, the highest number of Ephestia kuehniella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.     

Strain differences in the indian meal moth,Plodia interpunctella,in response to a granulosis virus

Three strains of the Indian Meal Moth,Plodia interpunctella, were compared in terms of their response to a granulosis virus under different environmental conditions. A significant difference in the relative susceptibilities to the virus of the three laboratory strains was established. Evidence of potential trade-offs with resistance was found in overall fecundity, pupal size and mortality at adult emergence. There was however little evidence that a reduction in resource level led to more trade-offs being apparent. No clear relationship was found between resistance to the lethal effects of the virus and susceptibility to the sublethal effects of the virus.     

Development of the larval ovary in the moth, Plodia interpunctella

The morphogenesis of ovaries and the organization of germ cells within them were visualized during the larval stages of the moth, Plodia interpunctella. The germ cells were observed by utilizing confocal microscopy coupled with immuno-fluorescent staining for the alpha-crystallin protein 25 (alphaCP25). The alphaCP25 was previously shown to be specific to germ cells of pupae and adults, and this study shows that alphaCP25 is present in larval germ cells as well. A cluster of 28 germ cells that stain for alphaCP25 was found in the gonads of newly hatched first instar larvae. The founding germ cells became segregated into four clusters, most likely by somatic cell intrusion, around the beginning of the second instar. Division of the primary germ cells began by the end of the second instar and the formation of all cystoblasts appeared to be completed within the four ovarioles by the end of the third instar. Within the ovarioles of third instar larvae, the germ cells were organized with a distal cap of seven germ cells which was segregated from the majority of the germ cells. The main body of germ cells was arranged around a central germ cell-free core as a spiral. Divisions of the cystoblasts to form cystocyte clusters were nearly completed during the fourth (last) larval instar. These features suggest that the strategy to produce follicles in moths is fundamentally different from the fruitfly, Drosophila. It appears that during the initial stages of ovary development in P. interpunctella, the primary germ cells undergo stage-complete divisions that are completed prior to the onset of the next set of divisions, which results in a complete complement of follicles available by the time of adult eclosion, while in Drosophila the primary germ cell divisions are initiated in the adult stage, and follicles are produced individually as resources are available.     

The effect of temperature and light pulses on the induction of diapause in the Toyama strain of the Indian meal moth, Plodia interpunctella

Abstract.The photoperiodic response in Plodia interpunctella collected at Toyama (36.7°N) was of long-day type and highly sensitive to temperature. The critical photoperiod giving 50% diapause was between 14 and 16 h at 20°C, between 12 and 14 h at 25°C and between 6 and 8 h at 30°C. Effects of night interruption by a 2-h light pulse on the diapause response were examined at 25°C on different background photoperiods ranging from LD 12:12 h to LD 2:22 h. Percentage diapause was very low when the middle portion of dark period was interrupted, so that U- or V-shaped response curves were obtained with background scotophases longer than 12 h. In these curves, the descending slopes were less steep than the ascending slopes. The critical dark period measured from dusk to an interrupting light pulse was about 1.5 h longer than the critical dark period ( c . 10 h) in the normal photoperiodic response. The critical dark period from the interrupting light pulse to dawn, on the other hand, was not parallel to dawn but shorter than the normal critical period in LD 12:12 h and LD 10:14 h and longer than that in LD 7:17 h to LD 4:20 h, indicating that the priming effects of the light pulse might be under the influence of the photophase.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in na?ve insects and in insects manipulated with bacterial and hormonal treatments.     

Upregulation of heat shock protein genes by envenomation of ectoparasitoid Bracon hebetor in larval host of Indian meal moth Plodia interpunctella

Effect of envenomation of ectoparasitoid Bracon hebetor was determined on the heart rate and the expression of shsp, hsc70 and hsp90 of the lepidopteran host Plodia interpunctella. Envenomated host larvae were promptly immobilized but heart rate was not changed until 4 days after envenomation. Northern hybridization showed that each hsp gene was differentially influenced by envenomation: continued high induction of shsp, gradual strong induction of hsc70, but no induction of hsp90. Our results suggest that upregulation of both shsp and hsc70 may produce potent factors that have important roles in the mechanism of host-parasitoid relationship.     

Observations on the intensity of diapause and cold tolerance in larvae from twelve populations and two reciprocal crosses of the Indian meal moth, Plodia interpunctella

ABSTRACT. The duration of diapause in larvae of Plodia interpunctella (Hübner) (Lepidoptera, Pyralidae) was assessed at 20°C in LD 11:13. Mean times from hatch to pupation for diapausing larvae from different populations ranged from 88 to 236 days. Most non-diapausing larvae pupated within 70 days at this temperature. Transferring diapausing larvae to 25°C and LD 9:15, or to 20°C and LD 15:9, 70 days after hatch reduced the subsequent mean time to pupation by 18–82% and 9–63% respectively. Only two population samples terminated diapause faster under LD 15:9 at 20°C than under LD 9:15 at 25°C. The mortality of diapausing larvae caused by 6- or 10-week exposures at 5, 7.5 or 10°C was generally less than 25%. Hybrids produced from a reciprocal cross between a temperate and a tropical African stock survived well. For other stocks there was some correlation between survival and diapause intensity. The low temperature regime which resulted in the greatest shortening of pupation time after return to the conditions used to induce diapause, did not always coincide with the temperature permitting the best survival. Results, however, indicate that some individuals of all stocks but one from the tropics are likely to survive in the U.K.     

Effect of four different nutrition regimes on the alpha-amylase gene expression in the Indian moth,Plodia interpunctella

Plodia interpunctella (Hübner 1813) (Lepidoptera: Pyralidae) is a cosmopolitan species known to infest a wide range of dried plant materials, including legumes and cereals. In this study, enzyme activity and expression of alpha-amylase gene in larval P. interpunctella grown on four different nutrition regimes was studied. The different nutrition regimes caused differential change in expression of alpha-amylase gene. Compared with the control (artificial diet), the walnut regime caused a 10.8% increase in expression of the alpha-amylase gene, whereas 79.3, 52.5, and 7.5% decreases were observed in the larvae grown on dates, raisins and oleaster respectively. The results also showed that glucose repression varied among larvae feeding on different food diets. The results revealed that expression of the alpha-amylase coding gene was significantly reduced in the larvae grown on date and raisin containing high amounts of glucose. A biochemical assay indicated overlapping of alpha-amylase activity and expression. Complexity in expression of amylase genes suggests the existence of a negative feedback mechanism in the insect larvae. Different parameters can affect gene expression and activation, which may explain the results presented here.     

Major digestive carbohydrase during larval development of meal moth, Plodia interpunctella (Lepidoptera: pyralidae)

The digestive system of P. interpunctella was characterized during its larval development to determination of carbohydrases using disaccharides (sucrose and maltose) and polysaccharides (starch and inulin) as substrate. At 6(th) instar larval, Invertase>alpha-amylase> maltase activities peaks were observed. Invertase was fractionated with acetone and isolated. The Invertase was 485.5 fold purified by Sephacryl S-200 and DEAE-Sephadex. Its kinetic parameters were K(m) of 6.6 mM, V(max) of 0.48, pH optimum of 5.5 and temperature optimum of 30 degrees C. This enzyme was activated by CaCl(2) and inhibited by EDTA. When analyzed by SDS-PAGE it showed one band of M(r) 34 kDa. The understanding of the digestive system of P. interpunctella could be a key step in the design of bioinsecticides.     

Factors influencing the duration and termination of diapause in the Indian-meal moth, Plodia interpunctella

The influence of environmental factors on the duration of diapause in Plodia interpunctella larvae reared in short photoperiods at 20 or 25° C was examined, Diapause terminated most rapidly in long photoperiods at high temperatures. Pupation was more delayed, and mortality was higher, in darkness than in the presence of light. At 20° C, LD 16: 8 hastened diapause termination only slightly in unchilled samples. Chilling for 10 weeks at 10° C greatly reduced the duration of diapause at 20 or 25° C in constant darkness, and rendered LD 16:8 effective in terminating diapause at 20° C. In addition, the quite short duration of diapause under LD 16:8 at 25° C was further shortened by holding for 6–10 weeks at 10° C or below, or by holding in an outbuilding during winter. Holding diapausing larvae at 15 or 20° C proved less effective. Temperature rises from 20 to 25 or 30° C proved effective in terminating diapause. In one stock, the temperature at which diapause was induced influenced its subsequent duration. Lighting conditions during induction had less influence on duration than had temperature, and no difference occurred between pupation times of larvae reared at different population densities, Under all conditions tested, diapause lasted longer in a recently collected field stock than in a laboratory stock.     

Regional differentiation of fat bodies in larvae of the indianmeal moth,Plodia interpunctella

Larvae of the Indianmeal moth, Plodia interpunctella, contain two morphologically distinct fat bodies. Tan-colored, highly tracheated fat body located posteriorly in the abdomen was the predominant fat body tissue during the early larval instars. White, sheet fat body located more anteriorly became the predominant type during the fifth (last) larval instar and eventually occupied most of the space of the hemocoel. Ultrastructural morphology of tan fat body showed the tissue to be composed of cells containing numerous, large, spherical mitochondria, with only few lipid, glycogen, or protein storage structures. In contrast, white fat body was composed of cells that in later larval stages had organelles typical of storage functions. Both fat bodies produced storage proteins during the late fifth instar, whereas only white fat body accumulated the storage proteins. Tan fat body dispersed and apparently autolyzed in pharate pupae, whereas the white fat body metamorphosed and persisted into the adult stage. These observations indicate that fat body of the Indianmeal moth is functionally and morphologically differentiated along the anterior-posterior axis into two regional subgroups of cells.