Mass production,infectivity, and field application studies with the entomogenous fungus Paecilomyces farinosus

Hyphal bodies (HB) of the entomogenous fungus Paecilomyces farinosus mass produced in shaken liquid cultures are infective per os and topically to Spodoptera exigua larvae. Pathogenicity of HB was greatest at pH 5.5. It was reduced by exposure for more than one-half hour to temperatures above 30°C. P. farinosus infected, killed, and produced mycelia on the surface of first-instar S. exigua larvae at a temperature—relative humidity combination as low at 15°C and 70% RH. Using two types of microdroplet applicators (droplet in the size range 1–30 and 30–90 μm, respectively) under field conditions, HB were delivered to distances of 76 m from the site of spraying. The number of HB deposited on the leaf surfaces of treated crops caused mortality of 70 to 90% in bioassayed larvae.     

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.     

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.     

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between ?12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (?12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (?12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C–40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.     

Catenaria anguillulae in the mermithid nematode Romanomermis culicivorax

Disease epizootics in laboratory cultures of the mermithid nematode Romanomermis culicivorax were caused by a chytridiomycetous fungal parasite, Catenaria anguillulae. Techniques used for mass rearing the nematode provided ideal conditions for dispersal of zoospores and multiplication of the fungus; up to 90% of the postparasites of R. culicivorax were parasitized. The fungus was isolated and grown on yeast extract-soluble starch agar, and the effects of different temperature and pH regimes on the fungus were investigated as possible methods for prevention or control. A temperature suitable for mass rearing the nematodes but unsuitable for the fungus could not be found, as C. anguillulae had a broad temperature range. Adjustment of pH or a short period of chilling (0°C) were more promising control measures. At acid pH (<5), zoospore motility and germination and infection of postparasites were suppressed. The disease was controlled by rearing nematodes in water adjusted to a pH of 4.5.     

The temperature and humidity preferences of Haemaphysalis longicornis,ixodes holocyclus and Rhipicephalus sanguineus (ixodidae): Studies on engorged larvae

The engorged larvae of Haemaphysalis longicornis, Ixodes holocyclus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to see whether the nature of their requirements in the laboratory were similar to the climate within the geographic range of each species. The response of H. longicornis and I. holocyclus to changes in humidity was also studied. Moulting of I. holocyclus larvae occurred from 18 to 28°C and at a saturation deficit of 4 mm Hg or less. The larvae of R. sanguineus moulted between 18 and 38°C and tolerated saturation deficits up to 35 mm Hg. The larvae of H. longicornis moulted between 15 and 38°C at saturation deficits up to 8 mm Hg. When engorged larvae of H. longicornis and I. holocyclus were exposed to very dry conditions for different periods of time and then transferred to moist conditions, the minimum pre-moult period and mortality was increased in comparison with larvae kept continuously under moist conditions. On the other hand, mortality of H. longicornis larvae was reduced in very dry conditions provided that they had been exposed to a moist environment for about 5 days previously. The larvae of I. holocyclus required at least 12 days exposure to a moist environment before any survived to moult in moderately dry conditions. The engorged larvae of both H. longicornis and I. holocyclus lost weight rapidly in dry air, whereas weight loss from R. sanguineus larvae was much slower. The rate of development was fastest in R. sanguineus and slowest in I. holocyclus.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

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.     

Hymenolepis diminuta (Cestoda): Effects of parasite density and temperature on the water balance of Tenebrio molitor and subsequent infectivity of the cysticercoids

The effects of the density of Hymenolepis diminuta and the effects of thermal acclimation on the water balance of Tenebrio molitor were examined. Also, the subsequent infectivity of the cysticercoids for rats were investigated. T. molitor beetles were fed known numbers of H. diminuta eggs and then were kept at 15° or 25°C for 14 days. After 14 days, beetles were desiccated and water loss was determined. Parasite density did not significantly affect transpiratory water loss in T. molitor kept at 15° or 25°C following 24 or 48 hr of desiccation. However, after 72 hr of desiccation, beetles maintained at 15°C evidently could not regulate water efficiently since there was a significant increase in the transpiratory water loss as parasite density increased. Beetles acclimated at 15°C produced fewer cysticercoids than did beetles maintained at 25°C. Also, fewer adult worms were recovered from rats intubated with cysticercoids from heavily infected, 15°C-acclimated beetles. Apparently, heavily infected beetles acclimated to 15°C do not produce viable cysticercoids.     

Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

The effect of temperature on phototaxis and geotaxis by larvae of the crab Rhithropanopeus harrisi (Gould)

Investigations of the effect of sudden temperature change on the phototaxis of Stage I and IV zoeae upon stimulation from horizontal and vertical directions with 500-nm light indicate a temperature-induced geotactic response in larvae of the crab Rhithropanopeus harrisi (Gould). For the horizontal tests both zoea stages were reared at 20 °C. Stage I showed positive phototaxis at temperatures between 15 ° and 35 °C, while Stage IV responded over the range of 10–30 °C. For the vertical tests, larvae, reared at 25 °C, were stimulated with overhead lights. Stage I zoeae ascended at 15 °, 20 ° and 25 °C and descended at 5 °, 10 °, 30 ° and 35 °C. Stage IV zoeae ascended at 20 ° and 25 °C and descended at 5 °, 10 °, 15 °, 30 ° and 35 °C. Although the descent at high temperatures could result from a negative phototaxis, a reversal in phototactic sign at high temperatures was not found in the horizontal experiments and the same vertical movement pattern is observed in total darkness. Upon exposure to high temperatures near the water surface, larvae would descend by means of a positive geotaxis rather than a negative phototaxis. This response involves active swimming by Stage IV larvae and passive sinking by Stage I.     

Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae)

The pathogenesis and midgut histopathology which resulted when larvae of the blackfly, Simulium vittatum, were exposed to Bacillus thuringiensis at various temperatures and periods of exposure were investigated. The onset of mortality was studied at 10°, 15°, 19°, and 24°C. For each 4–5°C increase in temperature above 15°C, the onset of mortality was shortened by 24 hr. Exposures as brief as 15 min to 10 ppm of a whole spore preparation resulted in an average mortality of 29% in late-instar larvae. Mortality increased sharply for exposures up to 3 hr, approaching a maximum of 80%.The gross signs of disease included cessation of feeding and tetany with brachytosis. The tissue most affected was the midgut epithelium in the regions of the gastric caeca and posterior stomach. The formation of cytoplasmic vacuoles followed by cell lysis and/or sloughing were very apparent in moribund larvae. Death resulted without bacteremia.     

Effects of temperature on hatching rate, embryonic development and early larval survival of the edible sea urchin, Tripneustes gratilla

The temperature tolerances of embryonic and early larval development stages of Tripneustes gratilla were investigated from 13-34°C under laboratory conditions. Zygotes showed unequal cleavage at 13°C, whereas cleavage did not occurred at 34°C. Hatching was observed between 16–31°C with maximum hatching rates observed at 22–29°C. The lower and higher temperature limits for embryonic development were approximately 22°C and 29°C, respectively. Outside of this temperature range, embryos showed abnormality at different incubation times. Early larvae of this species have the ability to survive the higher temperature limit for short periods of time. Prism and 2 arm pluteus larvae survived at temperatures between 30 and 33°C, whereas 4 arm pluteus larvae survived at temperatures between 30 and 36°C for 2 h. These results suggest that the larval temperature tolerance capability of T. gratilla is stage dependent. These findings are important for understanding the life history strategy of this sea urchin in the shallow open water environment.     

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.     

Cellular immune competence of a Drosophila mutant with neoplastic hematopoietic organs

In the Tum^l mutant of Drosophila melanogaster, the larval hematopoietic organs undergo neoplastic changes and release into circulation large numbers of blood cells. The lamellocytes, and to a lesser extent the plasmatocytes from which they are derived, are the cells that encapsulate various endogenous tissues and form melanotic tumors. The mutation is temperature sensitive, with maximum gene expression manifested at 29°C. The ability of Tum^l larvae to encapsulate eggs of the wasp parasite Leptopilina heterotoma is dependent not only on temperature, with host larvae much more immune reactive at 29°C than at lower temperatures (15° or 21°C), but also on the interval of time following infection when temperature shift experiments are performed. When the shift of parasitized larvae from 21° to 29°C is delayed by 18 hr the hosts are not as immune reactive as those shifted immediately after infection. Since Tum^l larvae are potentially highly immune reactive at the time of infection (with sufficient numbers of lamellocytes in circulation to encapsulate parasites), the low degree of immune competence in hosts shifted to 29°C after 18 hr or maintained at lower temperatures suggests that the increased capacity of blood cells to react against foreign surfaces is dependent on the cells acquiring new or altered recognition and adherence properties at 29°C. The 18-hr delay may provide the parasite with an opportunity to interfere with the acquisition of these specific cellular alterations. Differential hemocyte counts from parasitized larvae show abnormally low lamellocyte counts in susceptible hosts, indicating that successfully developing parasites interfere with the differentiation of hemocytes.     

Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1–6.5 on isoelectric focusing gel electrophoresis.     

Temperature and salinity tolerances of larval Californian grunion, Leuresthes tenuis (Ayres): A comparison with gulf grunion, L. Sardina (Jenkins & Evermann)

Temperature and salinity tolerances were determined for larval California grunion, Leuresthes tenuis (Ayres), and compared with previous data for Gulf of California grunion, L. sardina (Jenkins & Evermann). Larvae of similar age and acclimation history showed little interspecific difference in thermal tolerance, as measured by half-hour LT₅₀ values for 20–30 day old late postlarvae acclimated at various temperatures, and by upper and lower incipient lethal temperatures for 18°C-acclimated prolarvae. The upper incipient lethal temperature differed by 1 deg.-C (32°C for L. tenuis, 31°C for L. sardina), while the lower incipient lethal temperature for the 18°C acclimated prolarvae of both species was 7.5°C. L. tenuis larvae were much less euryhaline than L. sardina, with incipient lethal salinities of 4.2–41 %. for prolarvae and 8.6–38 %. for 20-day-old postlarvae; comparable values for L. sardina are 4–67.5 %. and 5–57.5 %. Both species show a decrease in temperature and salinity tolerance with age. The larvae of these disjunct congeners show a significant physiological divergence in euryhalinity but not in overall temperature tolerance. These tolerances are discussed in relation to the respective geographic ranges and behavioral responses of the two species.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

The influence of temperature, relative humidity and host factors on the attachment and survival of Boophilus microplus (Canestrini) larvae to skin slices

Attachment of B. microplus larvae was examined using slices (0.5 mm) of bovine skin stretched over a suitable medium. Optimal temperature for attachment lay between 31 and 38°C. At 38°C, 70 to 80% had attached by 4 h but this was followed 6–7 h after the release of the larvae by a significant decrease in the percentage attached. By 8 h, the percentage attached had risen again and remained at 70–80% for the subsequent 16 h. Attachment was not influenced by ambient relative humidity within the range 20–75 % on the time scale studied (16 h). Most larvae denied access to the skin surface at 20 or 45 % R.H. (38°C) died within 24 h. Those allowed to feed survived at 45% but most died at 20% R.H. There was no difference in attachment when bovine or rabbit serum or phosphate buffered saline was used, nor when skin was taken from Zebu × European cattle or Herefords. Attachment was no different when skin was taken from neck, rib or rump, or from cattle with different levels of resistance to B. microplus but there was reduced attachment with mouse skin. Attachment was reduced on skin which had been stored at ? 15°C for 1 day but storage at 10°C for 16 h had no effect. These results are related to problems of tick water balance, host specificity and stimuli for tick attachment.