Vertical transmission of Nosema fumiferanae (Microsporidia: Nosematidae) and consequences for distribution, post-diapause emergence and dispersal of second-instar larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

We examined vertical transmission of Nosema fumiferanae in the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae), and how it affects overwintering distribution and survival and spring emergence and dispersal of second-instar larvae in outbreak populations. Females containing 5.0 x 10(5) spores or more consistently produced 100% infected progeny. Transmission efficiency was still 50% at burdens as low as 0.2 x 10(5) spores per moth. Infection intensity in offspring increased with maternal spore load but became highly variable above 25 x 10(5) spores per female. Nosema multiplied in second instars for at least 1 month after they entered dormancy, regardless of temperature (2 degrees C versus 21 degrees C). Infection did not affect the distribution of overwintering larvae in a white spruce canopy. Dormancy survival between late-summer and the following spring was lower in families from infected females and was negatively correlated with larval infection intensity. Infection delayed larval emergence from hibernacula in the spring and resulted in delayed dispersal of emerged larvae, at least when parasite prevalence and infection intensities were high. Infected larvae were less successful in establishing feeding sites after dispersal. Our results underscore the potential of Nosema infection to negatively affect processes early in the budworm life cycle.     

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae)

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.     

Interactions between Nosema pyrausta (Microsporidia: Nosematidae) and Bacillus thuringiensis subsp. kurstaki in the European corn borer (Lepidoptera: Pyralidae).

Larval susceptibility to Bacillus thuringiensis was determined for Nosema pyrausta-infected and uninfected European corn borers, Ostrinia nubilalis (Hübner), in bioassays using a commercial formulation of B. thuringiensis subsp. kurstaki, Dipel ES, incorporated into diet. LC50 values for N. pyrausta-infected larvae were significantly lower (P<0.0001) than for uninfected larvae and declined with increasing levels of infection. LC50 values for a 15-d bioassay using field-colony first instars were 0.006 and 0.027 mg of Dipel ES/kg of diet for larvae moderately infected by N. pyrausta and uninfected larvae, respectively. Nosema pyrausta-infected larvae reared on Dipel ES-amended diets produced 70-fold fewer spores (P<0.0001) than larvae reared on standard diet. For example, 15 d after placement as first instars on standard diet, infected field-colony larvae produced 7.6-8.7 million N. pyrausta spores per larva; similar larvae placed on diet containing 0.09 mg of Dipel ES/kg of diet produced 85-103 thousand spores per larva. Infected larvae also weighed less and failed to mature on Dipel ES-amended diets. Increased susceptibility of N. pyrausta-infected larvae to Dipel ES and reduced N. pyrausta spore production in larvae feeding on diet containing Dipel ES suggest that Bt corn will have a direct adverse effect on the survival and continual impact of N, pyrausta as a regulating factor on European corn borer populations.     

Horizontal and vertical transmission of a Nosema sp. (Microsporidia) from Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae), a serious defoliator of deciduous trees, is an economically important pest when population densities are high. Outbreaking populations are, however, subject to some moderating influences in the form of entomopathogens, including several species of microsporidia. In this study, we conducted laboratory experiments to investigate the transmission of an unusual Nosema sp. isolated from L. dispar in Schweinfurt, Germany; this isolate infects only the silk glands and, to a lesser extent, Malpighian tubules of the larval host. The latent period ended between 8 and 15 days after oral inoculation and spores were continuously released in the feces of infected larvae until pupation. Exclusion of feces from the rearing cages resulted in a 58% decrease in horizontal transmission. The silk of only 2 of 25 infected larvae contained microsporidian spores. When larvae were exposed to silk that was artificially contaminated with Nosema sp., 5% became infected. No evidence was found for venereal or transovum (including transovarial) transmission of this parasite.     

Influence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microsporida: Amblyosporidae) and Aedes aegypti (Diptera: Culicidae).

Larvae of Aedes aegypti, transovarially infected with Edhazardia aedis, were reared between 20 and 36 degrees C to determine the influence of temperature on the development of the parasite and the infected host. Development of the parasite was evaluated based on spore yield and size. The predicted optimum temperature for maximum spore production of E. aedis in A. aegypti was 30.8 degrees C. The results demonstrate that the E. aedis-A. aegypti system has a wide temperature tolerance; whereas spore yield will be lower at unfavorable temperatures, the host will remain infected. Additionally, spores were significantly smaller from individual reared at 34 degrees C than those reared at either 20 or 27 degrees C. Development of the infected host was evaluated based on pupal weight and time of pupation. Infected pupae were significantly larger than uninfected pupae. There was also a significant difference in the pupation rate between controls and infected A. aegypti larvae. Controls had a 50% cumulative pupation time (CPT50) of 65.7 degree days and infected individuals a CPT50 of 76.6 degree days.     

Effect of temperature and relative humidity on the cellular defense response of Ephestia kuehniella larvae fed Bacillus thuringiensis

While maintained under all combinations of three temperatures and two RH, fifth instar larvae of the Mediterranean flour moth, Ephestia kuehniella were fed wheat treated with spores and crystals of Bacillus thuringiensis var. kurstaki. Larvae that had fed on wheat with the bacterial preparation contained higher concentrations of nodules in their haemocoel than did larvae fed on wheat without bacteria. Nodule concentrations in larvae fed untreated wheat were unaffected by temperature or relative humidity. Larvae fed treated wheat had higher nodule concentrations at 32 degrees C than at 15 and 23 degrees C, and higher nodule concentrations at a relative humidity of 85% than at 43%. The percentage of larvae that pupated was lower when larvae were fed the bacterial preparation, and was significantly higher at 23 degrees C than at 15 and 32 degrees C, regardless of whether larvae were fed bacteria or not. Less time was required for larvae to develop to pupation at higher temperatures and at higher humidity. Mean time to pupation was lower for bacteria-fed larvae than for those fed untreated wheat, and this appeared to be a result of truncation of the distribution of times to pupation because only rapidly developing larvae survived to pupation.     

Epizootiology of Hyalinocysta chapmani (Microsporidia: Thelohaniidae) infections in field populations of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae): a three-year investigation

The epizootiology, transmission dynamics and survival strategies employed by the microsporidium Hyalinocysta chapmani were examined in field populations of its primary mosquito host, Culiseta melanura and its intermediate copepod host, Orthocyclops modestus over a three-year period in an aquatic subterranean habitat. H. chapmani was enzootic and was maintained in a continuous cycle of horizontal transmission between each host. There were three distinct periods during the summer and fall when developing mosquito larvae acquired infections; each was preceded by or coincident with the detection of infected copepods. Results were corroborated in laboratory bioassays, wherein transmission was achieved in mosquito larvae that were reared in water and sediment samples taken from the site during the same time periods. The highest infection rates, ranging from 60% to 48%, were repeatedly observed during the first six weeks of larval development. These were coincident with the most sustained collections of infected copepods obtained during the year and highest levels of infection achieved in the laboratory transmission studies. The high prevalence rates of lethal infection observed in larval populations of C. melanura at this site are among the highest recorded for any mosquito-parasitic microsporidium and clearly suggest that H. chapmani is an important natural enemy of C. melanura. H. chapmani appears to overwinter in diapausing mosquito larvae but may also persist in copepods. The absence of vertical transmission in the life cycle of H. chapmani and the sole reliance on horizontal transmission via an intermediate host are unique survival strategies not seen among other mosquito-parasitic microsporidia. The epizootiological data suggest that this transmission strategy is a function of the biological attributes of the hosts and the comparatively stable environment in which they inhabit. The subterranean habitat is inundated with water throughout the year; copepods are omnipresent and C. melanura has overlapping broods. The spatial and temporal overlap of both hosts affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host. It is hypothesized that natural selection has favored the production of meiospores in female host mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Factors affecting the toxicity of Bacillus thuringiensis var. israelensis and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi (Diptera: Chironomidae)

Laboratory bioassays (48h duration, 25+/-1 degrees C) were used to determine the toxicity of Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi, a major pest of rice in southern Australia. Bioassays were conducted using different combinations of larval ages and densities to determine if these factors affected toxicity. The effects of temperature and substrate type on B.t.i. toxicity were also investigated. Tests were conducted using a commercial B.t.i. formulation (VectoBac WDG, 3000ITU/mg), a spore/crystal mixture derived from the VectoBac WDG strain, and VectoLex WDG, a commercial B. sphaericus formulation (650ITU/mg). VectoBac WDG was highly toxic to fourth instar C. tepperi in bioassays using a sand substrate (LC(50) 0.46mg/L, older larvae); younger fourth instar larvae were more susceptible (LC(50) 0.20mg/L). Increasing larval densities (from 10 to 30 per bioassay cup) increased LC(50) values for both age groups, significantly so in the case of older larvae (higher density LC(50) 0.80mg/L). Use of a soil substrate increased the LC(50) value (older larvae, 10 per cup) to 0.99mg/L. Similar differences in toxicity relative to larval age and substrate type were found in bioassays using the B.t.i. spore/crystal mixture. VectoBac WDG and the spore/crystal mixture both showed similar (approximately 6-fold) declines in activity between 30 and 17.5 degrees C. At lower temperatures (between 17.5 and 15 degrees C), activity of the spore/crystal mixture declined much more rapidly than that of VectoBac WDG. VectoLex WDG showed very low toxicity to C. tepperi larvae, and the overall impact of larval age and density was relatively minor (LC(50) values 1062-1340mg/L). Autoclaving VectoLex WDG did not substantially reduce its toxicity (LC(50) 1426mg/L), suggesting that formulation additives (i.e., surfactants and other adjuvants) are responsible for much of the toxicity occurring at the high product concentrations required to cause C. tepperi mortality. Whilst VectoLex WDG was ineffective against C. tepperi, VectoBac WDG has the potential to provide selective control of this rice pest at economically viable application rates.     

Microsporidian infections in Lymantria dispar larvae: interactions and effects of multiple species infections on pathogen horizontal transmission

The interactions in multiple species infections and effects on the horizontal transmission of three microsporidian species, Vairimorpha disparis, Nosema lymantriae and Endoreticulatus schubergi, infecting Lymantria dispar were evaluated in the laboratory. Simultaneous and sequential inoculations of host larvae were performed and the resulting infections were evaluated. Test larvae were exposed to the inoculated larvae to measure horizontal transmission. Dual species infections demonstrated interspecific competition between Nosema and Vairimorpha in the host larvae, but no observable competition occurred between Endoreticulatus and either of the other microsporidian species. Timing of inoculation was an important factor determining the outcome of competition between Nosema and Vairimorpha. The species inoculated first showed a higher rate of successful establishment; a time lag of 7 days between inoculations allowed the first species to essentially exclude the second. The microsporidia differed in efficiency of horizontal transmission. Nosema and Endoreticulatus were transmitted at very high rates, close to 100%. Horizontal transmission of Vairimorpha was less efficient, ranging from 25% to a maximum of 75%. The patterns of infection observed in inoculated larvae were reflected in the test larvae that acquired infections in the horizontal transmission experiments. Competition with Vairimorpha suppressed horizontal transmission of Nosema after simultaneous and sequential inoculation. In simultaneous inoculation experiments Endoreticulatus had no effect on transmission of Nosema and Vairimorpha.     

Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat

The epizootiology of Amblyospora camposi was studied in a natural population of Culex renatoi, a bromeliad-inhabiting mosquito, and its intermediate host, Paracyclops fimbriatus fimbriatus, over a 2-year period. Twenty Eryngium cabrerae plants were sampled monthly from January 2003 to January 2005 and the prevalence of A. camposi in P.f. fimbriatus and Cx. renatoi populations was determined. The monthly prevalence rates of meiospore infections in Cx. renatoi larvae never exceeded 5.5% and was detected in 50% of the monthly samples. Meiospores were available in plants over the course of the study at a mean concentration of 2 x 10(4) meiospores/ml. Within each plant the parasite was maintained by horizontal transmission. P.f. fimbriatus with vegetative stages and mature spores were found regularly in bromeliads suggesting efficient meiospore infectivity to field copepod populations. The mean concentration of spores from copepods found in plants was 8 x 10(2) spores/ml. Infections in copepods were detected in 54% of the monthly samples with a prevalence rate ranging from 0.55 to 17.4% and an overall average of 5.1%. Vegetative stages in fourth instar mosquito larvae (probably derived from the horizontal pathway via spores formed in copepods) were detected in 12.5% of the monthly samples with an overall prevalence rate of 1.1%. Infections in female and male adults were detected in 20.8% of the monthly samples with an overall average of 4.1% and 6.8%, respectively.     

Prevalence of Nosema sp. (Microsporidia: Nosematidae) during an outbreak of the jack pine budworm in Ontario

Microsporidia are believed to play little or no role in outbreaks of the jack pine budworm, Choristoneura pinuspinus Freeman (Lepidoptrera: Tortricidae), because the short duration (2–4 years) of those outbreaks may not permit significant build-up of the pathogen. We conducted the first survey of Nosema sp. (Microsporidia: Nosematidae) over the course of a recent jack pine budworm outbreak in Ontario. Between 2004 and 2010 the outbreak defoliated a cumulative total of 1.78 million ha. Microscopic examination of ∼15,000 overwintering larvae collected over 6 years in sites with densities of 3 larvae per branch or more revealed widespread occurrence of Nosema at generally high infection intensities. The pathogen was present in 69.5% of the 518 plots that were monitored. Prevalence of infection was generally low (below 40% in 84% of plots with infected larvae) but reached high levels (80–95%) locally and increased rapidly in most infestations within 1–2 years of onset. We hypothesize that the habit of early-instar larvae to feed on developing male flowers (pollen cones) after spring emergence is critical in allowing rapid build-up of Nosema by increasing efficiency of horizontal transmission (higher density of both infected larvae and egested spores). Nosema infection may contribute to the complexity of jack pine budworm outbreak patterns by affecting egg recruitment and early-instar survival at the stand level in concert with known effects of budworm-induced reductions in pollen cone production on those processes.     

Effects of a novel microsporidium on the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae)

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10⁵, 10⁶, 10⁷ spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10⁶ and 10⁷ spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.     

Sources of spores for the possible horizontal transmission of Thelohania solenopsae (Microspora: Thelohaniidae) in the red imported fire ants, Solenopsis invicta

We screened adult and larval secretions and midden piles for the presence of Thelohania solenopsae spores to decipher potential sources for the horizontal transmission of the pathogen in fire ants. Hemolymph samples from both adult and larvae were also screened to rule out hemolymph contamination of samples. In adults, Thelohania spores were found in the crop and the fecal fluids, although only free spores were found in the fecal fluids of adults. In fourth instar larvae, both free and octospores were seen in midgut and the meconium samples. All of the midden pile samples had T. solenopsae spores of both types. Based on these results, we theorize that the pathogen may be horizontally transmitted within a colony by the removal and sharing of meconium of prepupating fourth instar larvae by adult workers and by the adult fecal droppings, and intercolonially by contamination of midden piles or brood raiding.     

Specific and sensitive detection of Nosema bombi (Microsporidia: Nosematidae) in bumble bees (Bombus spp.; Hymenoptera: Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10(7) down to 10 spores diluted in 100 microl of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of approximately 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.     

Response of Lymantria dispar L. (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki at different ingested doses and temperatures

We examined mortality and feeding inhibition response of Lymantria dispar L. (Lepidoptera: Lymantriidae) larvae to ingested doses of Bacillus thuringiensis subsp. kurstaki as a function of dose, instar and temperature. We developed generalized (logistic) linear mixed models and a mixture survival model, commonly used in medical statistics, to analyze the complex data set. We conducted bioassays of Foray 48B with larvae from the NJSS laboratory stock, using droplet imbibing or force-feeding to ensure dose ingestion. The dose causing mortality in 50% of the test population (LD₅₀) under standard test conditions (22 °C) ranged from 0.019 International Units (IU)/larva for first instar larvae (L₁) to 1.6 IU/larva for L₄. Temperature affected larval mortality in two ways. Mortality occurred sooner and progressed more rapidly with increasing temperature (13-25 °C) at each dose level and instar, while the maximum level of mortality attained by each instar decreased with increasing rearing temperature. The mechanisms underlying this effect are being investigated. Larvae that survived exposure to B. thuringiensis resumed feeding after a period that was dependent on instar, dose, and temperature. The equations describing observed mortality and feeding recovery responses were used to construct a simulation model, which was able to predict both processes, and which forms the basis for a process-oriented model that can be used as a decision support tool in aerial sprays.     

Longevity and germination of Edhazardia aedis (Microspora: Amblyosporidae) spores

Edhazardia aedis is a polymorphic microsporidium of mosquitoes that is both horizontally and vertically transmitted to its host. Because it is being developed for biological control of mosquitoes, detailed knowledge is needed regarding the longevity and germation of its fragile, mosquito‐infectious spore. Spores responsible for horizontal transmission were extracted from 7–10‐day‐old larvae (reared from infected Aedes aegypti eggs) and purified by Ludox density gradient centrifugation. Mature spores were variable in specific gravity, being found throughout the 20 and 60% zone in Ludox gradients. The optimal environment for spore germination was dilute KCl at pH 10.0–11.0; ammonia inhibited germination. Osmotic inhibition was almost complete in both sucrose and polyethylene glycol at concentrations equivalent to 40 atm. The spores retained their viability for a maximum of 21 days at 23±2°C, whereas when held at 5±2°C, their viability was completely lost within two days post‐harvest. Potential for germination decreased along with infectivity, providing a simple assay for spore viability.     

Effect of soil temperature and moisture on survival and infectivity of Metarhizium anisopliae to four tephritid fruit fly puparia

The infectivity of 4 isolates of Metarhizium anisopliae to puparia of Ceratitis capitata treated as late third-instar larvae in unsterilized soil was investigated in the laboratory under controlled temperature and moisture. At 20-30 degrees C, mortality in puparia was highest at water potential of -0.1 and -0.01 mega Pascal (MPa) and lowest at water potential of -0.0055 and -0.0035 MPa in all the isolates. In wetter soil however, isolates ICIPE 20 and 60 caused significantly higher mortality than ICIPE 18 and 69. The survival of conidia in drier soil (-0.1 MPa) was not adversely affected at all temperatures. However, in wet soil (-0.0035 MPa) there was drastic reduction in colony counts in ICIPE 18 and 69 at 25 and 30 degrees C but conidial density in ICIPE 20 and 60 remained at the initial level at 14 days after inoculation at all temperatures. When ICIPE 20 was evaluated against three other fruit fly species (Ceratitis cosyra, Ceratitis rosa, and Ceratitis fasciventris), significant reduction in adult emergence and higher pupal mortality occurred in C. cosyra and C. fasciventris than in C. rosa at a combination of 15 and 20 degrees C and -0.1 and -0.0035 MPa. However, at higher temperature and the same moisture level, the isolates were equally pathogenic across the 3 species. It is probable that in addition to pathogen cycling and multiplication from dead infected insects in the soil, a balance between microbial degradation and replenishment of inoculum of virulent isolates occur through fluctuations in, and intricate interactions between temperature and moisture levels. This study is indicative of the potential of using isolate ICIPE 20 for soil inoculation against pupariating third-instar larva of fruit flies, thus providing a novel alternative to chemical soil application.     

Temperature and food quality effects on growth,consumption and post-ingestive utilization efficiencies of the forest tent caterpillar Malacosoma disstria (Lepidoptera: Lasiocampidae)

Temperature and food quality can both influence growth rates, consumption rates, utilization efficiencies and developmental time of herbivorous insects. Gravimetric analyses were conducted during two consecutive years to assess the effects of temperature and food quality on fourth instar larvae of the forest tent caterpillar Malacosoma disstria Hübner. Larvae were reared in the laboratory at three different temperatures (18, 24 and 30 degrees C) and on two types of diet; leaves of sugar maple trees Acer saccharum Marsh. located at the forest edge (sun-exposed leaves) or within the forest interior (shade-exposed leaves). In general, larvae reared at 18 degrees C had lower growth rates and lower consumption rates than larvae reared at the warmer temperatures (24 and 30 degrees C). Moreover, the duration of the instar decreased significantly with increasing temperatures. Type of diet also affected the growth rates and amount of food ingested by larvae but did not affect the duration of the instar. Larvae fed sun-exposed leaves consumed more food and gained higher biomasses. Values of approximate digestibility and efficiency of conversion of ingested food were also higher when larvae were fed sun-exposed leaves. Higher growth rates with increasing temperatures were primarily the result of the shorter stadium duration. The higher growth rates of larvae fed sun-exposed leaves were possibly the result of stimulatory feeding and consequently greater food intake and also a more efficient use of food ingested. This study suggests that the performance of M. disstria caterpillars could be enhanced by warmer temperatures and higher leaf quality.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.