Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Effect of dose of cytoplasmic polyhedrosis virus on infection,mortality, development rate,and larval and pupal weights of the pink bollworm

Host-pathogen relationships were studied between the pink bollworm, Pectinophora gossypiella, and a cytoplasmic polyhedrosis virus (CPV). Results showed that the median effective dose (ED₅₀), the dose that infects half the test subjects, was 1.91 × 10² polyhedral inclusion bodies (PIB)/ml of diet. The median lethal dose (LD₅₀) was 1.72 × 10⁵ PIB/ml. Diagnosis for CPV infection was more reliable in adult pink bollworms than in late-instar larvae. Duration of the larval stage increased with viral dose, but duration of the pupal stage was not affected by CPV. Weights of infected male and female pupae were 23.7 and 24.0% less than those of untreated pupae, respectively. Pupal weights were not significantly influenced by increases in the viral dose. Weights of larvae of a given age decreased as dose increased. The effect of CPV on duration of the immature stages and on pupal weight was not significantly influenced by rearing temperatures between 25.0° and 32.5°C. Pupal weight of infected pink bollworms decreased as the duration of the larval stage increased.     

Sclerotization as a long-term preservation method for Rosellinia necatrix strains

This work describes a simple protocol for longterm preservation of strains of Rosellinia necatrix based on sclerotia production combined with storage at 4°C in liquid substrate, without affecting the growth and pathogenic characteristics of the fungal isolates recovered. The sclerotization process was set up in both liquid and solid media, and the sclerotia-like structures (pseudosclerotia) obtained were preserved in liquid media or water at 4°C. R. necatrix pseudosclerotia viability after 6 years of preservation at 4°C was confirmed by growth and microscopic characteristics, with no differences when compared with the fungal strains routinely preserved by periodic transfers. Additionally, pathogenicity on avocado plants by the preserved R. necatrix strains showed no difference from those preserved by periodic transfers. The albino strain used in this study should continue to be preserved by periodic subculturing.     

Seasonal adaptations of different geographical populations of the sunflower moth, Homoeosoma electellum

A photothermogram constructed for a central Missouri population (ca. 39°N latitude) of the sunflower moth, Homoeosoma electellum (Hulst) predicted that this population has a seasonal life cycle of 2 complete and a partial third generation per year, and that daylengths of less than 13 h 30 min light/day and mean temperatures of less than 20°C induce the mature larval diapause. A test of the predicted life cycle revealed that larvae entered diapause when they were exposed from their first instar onwards to natural conditions in central Missouri beginning September 15, 1982. Sunflower moths obtained from northwest Texas (ca. 35°N latitude) and northeast South Dakota (ca. 45°N latitude) displayed shorter critical photoperiods for diapause induction at 20°C (12 h 30 min light/day and 12 h 15 min light/day, respectively) than did those from Missouri. The population of the sunflower moth obtained from South Dakota does not, therefore, appear to be adapted to local conditions, and moths might disperse from lower latitudes to establish transiet populations each year.     

Trans-ovum transmission of a cytoplasmic polyhedrosis virus of Heliothis virescens (Lepidoptera: Noctuidae)

Adults of Heliothis virescens infected with a cytoplasmic polyhedrosis virus (CPV) produced healthy offspring when their eggs were surface sterilized with either 15% formaldehyde or 0.2% sodium hypochlorite solution. Larvae from infected parents (1) cultured on a vitamin-deficient medium, (2) exposed to cold treatment (5°C, 24 hr), or (3) as progeny of adults from diapaused infected pupae, produced the same number of infected individuals as larvae reared in the customary way. Field studies indicated that the percent of CPV infection in larvae originating from virus-infected parents was density dependent.     

Enzyme cytochemical observations on the tissue stages of the life cycle of Eimeria acervulina and Eimeria necatrix

Cytochemical studies concerning the presence and distribution of various enzymes in the tissue stages of the life cycle of Eimeria acervulina and E. necatrix have been undertaken.Acid and alkaline phosphatases as well as ATPase (pH 7·2) were found to be present in all the stages of the life cycles examined, the latter enzyme being very strong in the plastic granules of the macrogametocytes and in the inner membrane of the oocyst wall. The reaction for all three enzymes was observed in the cytoplasm but not the nucleus. Non-specific esterase and glucose-6-phosphatase were also found in all stages examined, the latter occurring in greater amounts in stages where deposition of glycogen (amylopectin) was pronounced. Succinic dehydrogenase occurred only in the second generation schizonts and merozoites of E. necatrix and in the formed oocysts of both species. No reaction for β-glucuronidase or leucine naphthylamidase was apparent in any stage, although a slight reaction for leucine naphthlyamidase was seen in the second generation merozoites of E. necatrix lying free in the intestinal lumen.The presence and distribution of these enzymes and their possible function were discussed.     

Life cycle and behavior of Amblyomma rotundatum (Acari: Ixodidae) under laboratory conditions and remarks on parasitism of toads in Brazil

The life cycle and behavior of Amblyomma rotundatum were evaluated under laboratory conditions. The experiment started with four engorged females collected from toads (Rhinella schneideri) naturally infested at the Pirapitinga Ecological Station in the state of Minas Gerais, Brazil. Developmental periods of free-living stages were assessed in an incubator at 27 ± 1 °C, >80 % RH and darkness. The complete life cycle, including pre-attachment periods for each parasitic stage, ranged from 126 to 228 days. The pre-attachment, feeding and molting periods increased as the life cycle progressed from larva to adult female. Oviposition lasted about 20 days, with the peak occurring on days 4 and 5. Longevity of nymphs and adult females was quite similar (approximately 250 and 240 days, respectively) and slightly longer than that of larvae. Lesions caused by tick feeding are discussed and a list of known hosts, including new host records for A. rotundatum, is offered.     

Temperature-induced changes in the life cycle of Leuctra nigra(Plecoptera: Leuctridae) from a Lake District stream

1. The life cycle of Leuctra nigra (Olivier) took 2 years in a small stream in the English Lake District and the exponential growth of the larvae was scarcely affected by variations in water temperature (range 4.2-14.0°C). Mean growth rates for three year-classes were 0.43±0.01, 0.42±0.01, 0.39±0.05% body length day^?1. There were thirteen or fourteen larval instars for males and fourteen or fifteen for females. The ratio between successive instars was a constant 1.20 (conformed with Dyar's rule). 2. Larval growth and mortality were exponential at six constant temperatures (5.9, 8.2, 12.1, 15.8, 18.2, 19.8°C) in the laboratory. Mean growth rates (% body length day^?1) increased directly with temperature from 0.37 (5.9°C) to 0.55 (19.8°C). Mean mortality rates (% day^?1) increased directly with temperature from 0.20 (5.9°C) to 0.26 (12.1°C) and then markedly increased to 0.54-0.58 at the three higher temperatures. Only 7-10% of animals completed their life cycle at the three higher temperatures compared with 23–27% at the three lower temperatures. Egg production also decreased considerably at the higher temperatures. 3. As growth rates in the stream and laboratory were similar at similar temperatures (<14°C), the optimum conditions for growth in the laboratory were probably similar to those in the stream; therefore resources such as food and space were not restricting growth in the stream. 4. The implications of the temperature-induced changes in growth and mortality are discussed and it is concluded that although the life cycle can change from semivoltine to univoltine with increasing temperature, the costs of a univoltine life cycle are high in terms of survival and egg production, both of which decreased markedly between 12.1 and 15.8°C. Therefore the optimum habitat for this species appears to be a summer cool stream (maximum temperature <14°C) and the optimal life cycle appears to be about 2 years from egg to adult.     

Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures

Dalgliesh R. J. and Stewart N. P. 1979. Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures. International Journal for Parasitology9: 115–120. The temperature of incubation of unfed Boophilus microplus larvae infected with Babesia bovis influenced the morphology and infectivity of the Babesia within the tick. Incubation at 37°C for 1–3 days stimulated the development of parasites morphologically similar to those usually observed in fed larvae harvested from cattle; similar forms appeared more slowly in larvae incubated at 31°C or 25°C. Extracts prepared from larvae after incubation at 37°C for 3–5 days or 30°C for 8 days were consistently infective for cattle. Prior storage of larvae at 14°C for up to 28 days enhanced the development of infectivity at 37°C; infectivity could still be produced after 65 days storage at 14°C but not after 76 days. Larvae released on a host transmitted B. bovis sooner if they had been incubated at 37°C for 4 days. It was concluded that the development of B. bovis to an infective stage in B. microplus is temperature dependent and does not require the stimulus of feeding by the host.     

The ontogeny of the oribatid mite <Emphasis Type="Italic">Nanhermannia coronata</Emphasis> (Acari,Oribatida, Nanhermanniidae)

The ontogeny of the oribatid mite Nanhermannia coronata (Oribatida, Nanhermanniidae) was studied in the laboratory. At a temperature of 22–23°C and air humidity 100%, the entire life cycle from egg to adult lasted for 105–124 days. N. coronata juveniles and females are described. The diagnostic characters of the chaetom of N. coronata and N. nana larvae and nymphs are given. Morphological differences between N. coronata and N. nana allow distinguishing these species at all active stages.     

Selection for an optimal monovoltine life cycle in an unpredictable environment. Studies on the beetleCatops nigricans Spence (Col., Catopidae)

Summary Catops nigricans reproduces in the autumn. Pre-imaginal development is temperature-dependent and takes place during the winter, followed by aestivation in the early adult stage. This summer diapause is obligatory and temperature independent. It synchronizes the monovoltine life cycle with the annual cycle. In three populations collected near Kiel (54°22′ N, 10°6′ E), K?ln (50°54′ N, 7°6′ E), and Paris (49°25′ N, 2°20′ E), pre-imaginal development slowed and the duration of summer diapause decreased with increasing latitude. Synchronization of the critical breeding interval with the appropriate environmental conditions was achieved through temperature- and photoperiod-dependent sensitivity of ovipositing adults, through different thermal thresholds in eggs, larvae, and pupae, and through sensitivity to photoperiod in third-instars larvae.C. nigricans copes with the unpredictability of climatic conditions in different ways. The local populations have evolved a mean diapause length which probably adjusts the life cycle in most years to the optimal date for reproduction. The mean diapause length was 77 days for Kiel, 98 days for K?ln and 138 days for Paris at 10°C, short-day (=SD).C. nigricans also spreads the risk by varying diapause length. Amongthe progeny of single females the range of diapause duration covered about 70% of the total range of the whole population. The oviposition rate of females confined to subterranean life was the same as females confined to subterranean life was the same as in those living under the influence of a varying photoperiod.C. nigricans should therefore be able to live both in the litter layer of forests and also in the nests and galleries of small mammals.     

Some effects of time,temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae

Dalgliesh R. J. and Stewart N. P. 1982. Some effects of time, temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae. International Journal for Parasitology12: 323–326. Percentages of larval ticks in which Babesia bovis and B. bigemina parasites could be detected (infection rates) were determined after the larvae had been exposed to temperatures between 9°C and 27°C for periods of 1–35 days and then either fed on calves or heated at 37°C to stimulate babesial development. Infection rates with both species increased during 2–4 weeks after the larvae hatched, regardless of the temperature of exposure. Infection rates with B. bovis were higher after exposure of larvae to 14°C than to 27°C. This effect was less pronounced with B. bigemina. Infection rates were higher in fed larvae than in unfed, ‘heat stimulated’ larvae. The findings indicate that infected larval ticks become more efficient vectors of Babesia during the first 2–4 weeks after hatching and that repeated sampling of a tick population is necessary to determine valid infection rates.     

Development,metamorphosis, and natural history of the nudibranch Doridella obscura Verrill (Corambidae: Opisthobranchia)

The doridacean nudibranch Doridella obscura Verrill was raised through one complete generation in laboratory culture, and spawning behavior monitored for a year at monthly intervals in Barnegat Bay, New Jersey.The nudibranch deposited egg masses throughout the year in Barnegat Bay, and the larvae remained viable at temperatures ranging from 1.5 to 28 °C. At 25 °C the eggs hatch 4 days after oviposition, and the planktotrophic veliger larvae swim and feed for 9 days before they metamorphose. Settlement occurs specifically on the bryozoan Electro crustulenta (Pallas). The spirally coiled larval shell grows rapidly until the dorsal mantle fold is retracted from the aperture 5–6 days after hatching. Although starved larvae grow only slightly and do not metamorphose, they resume normal development on introduction of suitable food. Newly metamorphosed juveniles consume algae and debris on the surface of the bryozoan until they grow large enough to attack the living zooids of E. crustulenta.The life cycle of Doridella obscura is short (26 days at 25 °C), allowing the nudibranchs to take advantage of short-lived Electra crustulenta colonies in unstable habitats in bays and estuaries.     

Alterations in the production of hemocytes due to a neoplastic mutation of Drosophila melanogaster

The hemocytes of a genetically induced, temperature-sensitive lethal mutation of Drosophila, Tum¹, were examined both quantitatively and qualitatively during the third larval instar. At the tumor-permissive temperature, 29°C, there was a fourfold increase in the concentration of circulating hemocytes in mutant larvae as compared to control. Additionally, the relative frequency of lamellocytes was 30 times greater in Tum¹ larvae than Basc in the early third instar. However, the severity of this abnormality gradually diminished as Tum¹ approached pupariation; though high frequencies of lamellocytes were always present. At the tumor-restrictive temperature (15°C) the concentration of circulating hemocytes was over twice that found at 29°C for Tum¹ larvae, and did not change during the course of third instar. However, in contrast to 29°C there was no abnormal increase in the frequency of lamellocytes at the tumor-restrictive temperature. Control larvae had equivalent concentrations of hemocytes at both temperatures. In one of two temperature shift experiments, Tum¹ larvae shifted from 15° to 29°C at the beginning of third instar expressed the abnormal hemocyte concentration and differentiation associated with larvae raised continuously at 29°C. In addition, Tum¹ larvae shifted from 29° to 15°C expressed reduced abnormalities of hemocyte differentiation, e.g., with fewer lamellocytes in circulation. The possibility of a temperature-sensitive period for the activation of the Tum¹ gene is discussed.     

The influence of temperature on growth rate and length of larval life of the gastropod, Crepidula plana Say

The relationship between rate of larval development and the potential to prolong larval life was examined for larvae of the marine prosobranch gastropod Crepidula plana Say. Larvae were maintained in clean glass dishes at constant temperatures ranging from 12–29°C and fed either Isochrysis galbana Parke (ISO) or a Tahitian strain of Isochrysis species (T-ISO). Under all conditions, larvae grew at constant rates, as determined by measurements of shell length and tissue biomass. Most larvae eventually underwent spontaneous metamorphosis. Regardless of temperature, faster growth was associated with a shorter planktonic stage prior to spontaneous metamorphosis. Within an experiment, higher temperatures generally accelerated growth rates and reduced the number of days from hatching to spontaneous metamorphosis. However, growth rates were independent of temperature for larvae fed ISO at 25 and 29°C and for larvae fed T-ISO at 20 and 25°C. Where growth rates were unaffected by temperature, time to spontaneous metamorphosis was similarly unaffected. Maximum durations of larval life at a given temperature were shorter for larvae of Crepidula plana than for those of the congener C. fornicata (L.), although both species grew at comparable rates. Interpretations of the ecological significance of these interspecific differences in delay capabilities will require additional data on adult distributions and larval dispersal patterns in the field.     

Environmental induction of larval diapause and life-history consequences of post-diapause development in the Large Copper butterfly,Lycaena dispar (Lepidoptera: Lycaenidae)

Many insects in temperate zones withstand the adverse conditions of winter through entering diapause and the two most important environmental stimuli that induce diapause are photoperiod and ambient temperature. The Large Copper butterfly, Lycaena dispar Haworth (Lepidoptera: Lycaenidae), is a Palearctic butterfly that hibernates as larvae. Since this butterfly is a near threatened species in some regions, there has been a growing need for a standardized protocol for mass rearing of this butterfly based on the adequate knowledge of its ecology. In the present study, we first identified that L. dispar larvae were sensitive to the photoperiodic induction of diapause during their first larval instar. We then investigated to what extent the diapause-inducing effects of photoperiod could be modified by ambient temperatures in L. dispar larvae by exposing them to the range of day-lengths (L:D 14:10, 12:12, 10:14 and 8:16) at three different temperatures (15, 20 and 25 °C). All larvae were induced to enter diapause at low ambient temperature (15 °C) regardless of photoperiod, whereas most of them (86 %) exhibited direct development when temperature was high (25 °C). The photoperiodic induction of diapause was evident when day-length was shorter than 14 h at intermediate temperature (20 °C). Pre-diapause development was prolonged at low temperatures. Finally, we found that post-diapause development of L. dispar larvae was determined by both the chilling temperature experienced by diapausing larvae and the duration of larval diapause. Adult emergence was enhanced when larvae were chilled at 8 °C and when they had been under the state of diapause for 20 days before they were treated to terminate diapause.     

Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumoso-roseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and Spodoptera littoralis

Bioassays were conducted to determine the susceptibility of egg masses of Mamestra brassicae and Spodoptera littoralis to different spore doses of Paecilomyces fumoso-roseus and Nomuraea rileyi at 20° and 25°C. P. fumoso-roseus was highly virulent against eggs, whereas N. rileyi provoked only a deferred mortality of larvae hatched from treated eggs. Nevertheless, larval mortality of S. littoralis caused by N. rileyi at 25°C was more effective after first-instar larval contamination than after egg mass treatment. The duration of the egg stage could explain differences of susceptibility between the two noctuids at 25°C. Scanning electron microscopical observations suggested two ways of contamination of newly hatched larvae. First, fungal germinations on the chorion surface suggested that newly hatched larvae might be infected by penetration of the egg integument before hatching. Second, conidia on the egg cuticle could be an entomopathogenic inoculum for newly emerging larvae which fed upon chorions. Results showed that pathogenicity of Hyphomycetes to noctuid eggs might be a promising area of investigation for biological control.     

Effect of low rearing temperature on development of Galleria mellonella larvae and their sensitivity to juvenilizing treatment

• 1.1. The development of Gallena mellonella is strongly affected by a low temperature of 18°C (the last instar persists for more than one year, instead of about 9 days at 30°C). At 18°C the last instar Galleria mellonella larvae respond to juvenilizing treatment—chilling stress or juvenile hormone analogue—with a very low percentage or no supernumerary moults, respectively.
• 2.3. Experiments in which larvae subjected to such treatments were transferred from 18°C to 30°C and vice versa showed that for the realization of the larval programme after chilling stress application the higher (30°C) temperature is needed.
• 3.4. In last instar larvae reared at 18°C there coexist very high juvenile hormone titre and high juvenile hormone esterase activity.
• 4.5. This phenomenon which is found in both, chilled and unchilled larvae, is discussed.