The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

A method for isolating milky disease, Bacillus popilliae var. rhopaea, spores from the soil

A method based on the tyndallization procedure is described for isolation of Bacillus popilliae var. rhopaea spores from the soil. A soil suspension is diluted with a germinating medium, which promotes the germination of most spores except B. popilliae var. rhopaea, and is treated with a series of seven heat shocks (70°C for 20 min) at hourly intervals. This treatment reduced the number of contaminant spores by over 95%. The suspension is then plated out onto “J” medium which allows the germination and growth of all surviving spores including the milky disease spores. The plates are incubated anaerobically at 28°C for 7 days before the characteristic small transparent colonies of B. popilliae var. rhopaea are counted. In testing the method it was revealed that about 15% of the milky disease spores in the soil produced visible colonies, and that a spore concentration of over 1.2 × 10⁵ spores/g dry wt of soil could be quantified. This concentration of spores produces only 3% infection in Rhopaea verreauxi larvae. The method may be applicable to other varieties of B. popilliae which will grow on “J” medium.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

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

Susceptibility of Heliothis zea larvae to Nomuraea rileyi at various temperatures

Laboratory studies were conducted to determine the susceptibility of various larval instars of Heliothis zea to different spore doses of Nomuraea rileyi at constant and variable temperatures. The fungus was most effective at 20° and 25°C, with a mortality of 80% and 71%, respectively. At 15°C the disease progressed very slowly with larval mortality occurring in 12–28 days post-treatment. Conversely, at temperature ranges above 15°C, the mortality of the larvae occurred in 6–12 days. Three different combinations of variable temperatures included 20–30°, 25–30°, and 20–35°C, but mortality did not exceed 46%. Larvae in the third to fifth instars were more susceptible to infection than were those in the first and second instars.     

Influence of different temperatures on the tachinid parastite,Sturmiopsis inferens [Dip.]

The influence of constant temperatures of 27, 29, 31 and 33°C and alternating temperature of 31/33°C (18/6 h) onSturmiopsis inferens Townsend was studied during 12 successive generations. The larval and pupal periods for male parasites were 13.5±0.5 and 11.0±0.3 days respectively and for female 12.8±0.5 and 11.1±0.3 days respectively in the 1st generatioin at 27°C. It decreased progressively with increase in temperature. Survival of females, fertility and fecundity were adversely affected at higher temperatures. A temperature range of 27–29°C appeared to be optimum for mass rearing of the parasite in the laboratory. The higher premature mortality observed at a constant 33°C was not observed at temperatures fluctuating between 31/33°C. Presumably under field conditions, where temperature is constantly fluctuating, the flies will be able to withstand a comparatively higher temperature.     

Induction of larval diapause in the blowfly, Calliphora vicina R.-D. (Diptera, Calliphoridae) under field and laboratory conditions

Field experiments conducted in the environs of St. Petersburg (Russia) with a local population of Calliphora vicina showed that induction of larval diapause under natural conditions was significantly dependent on day lengths and temperature. The maternal photoperiodic response had a distinct threshold: the first diapausing larvae hatched from the eggs laid in the middle of August when the day length was 16 h; at shorter photoperiods, the fraction of diapausing larvae depended only on temperature. At the mean temperature of 16°C, larval diapause was rarely recorded; at 12–13°C, about 50% of the larvae entered diapause; at 7–9°C, nearly all the larvae entered diapause. These results of the field experiments agree well with the parameters of photoperiodic and thermal responses studied in the laboratory at constant temperatures and photoperiods.     

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.     

Germination behaviour of Conyza bonariensis to constant and alternating temperatures across different populations

Conyza bonariensis is one of the most problematic weed species throughout the world. It is considered highly noxious due to its interference with human activities, and especially the competition it poses with economically important crops. This research investigated the temperature requirements for seed germination of four populations of C. bonariensis with distinct origin and the influence of daily alternating temperatures. For this, a set of germination tests were performed in growth chambers to explore the effect of constant and alternating temperatures. Seeds of the four populations (from Lleida, Badajoz and Seville, Spain and Bahía Blanca, Argentina) were maintained at constant temperatures ranging from 5 to 35°C. The final germination and cardinal temperatures (base, optimum and maximum) of each population were obtained. We also tested the influence of daily alternating temperatures on final germination. To do so, seeds were exposed to two temperature regimes: 5/15, 10/20, 15/25, 20/30 and 25/35°C night/day temperature (intervals increasing 5°C, with constant oscillation of 10°C) and to 18/22, 16/24, 14/26, 12/28 and 10/30°C night/day temperature (intervals with average of 20°C, but increasing the oscillation in 4°C between intervals). In general, all populations behaved similarly, with the highest germination percentages occurring in the optimum temperature range (between 21.7°C and 22.3°C) for both constant and alternating temperatures. In general, climatic origin affected germination response, where seeds obtained from the coldest origin exhibited the highest germination percentage at the lowest temperature assayed. In addition, we observed that the alternating temperatures can positively affect total germination, especially in oscillations that were further from the average optimum temperature (20°C), with high germination percentage for the oscillations of 15/25, 20/30, 18/22, 16/24, 14/26, 12/28 and 10/30°C in all populations. The cardinal temperatures obtained were significantly different across the populations. These results provide information that will facilitate a better understanding of the behaviour of Conyza and improve current field emergence models.     

Interactive effects of rutin and constant versus alternating temperatures on performance ofManduca sexta caterpillars

The effects of different concentrations of rutin and constant temperature (20 °C) versus alternating temperatures (23∶15 °C) on growth, molting and food utilization efficiencies of third instar tobacco hornworms (Manduca sexta) were determined. Relative consumption rate (RCR) and relative growth rate (RGR) were significantly higher for larvae at the alternating thermal regime compared to those at the constant (representing the average) temperature. With increasing concentrations of rutin, the negative effect of rutin on RCR and RGR increased for the larvae in the alternating thermal regime; however, at the constant temperature, rutin had little effect. The alternating thermal regime promoted synchrony in the timing of spiracle apolysis (the earliest morphological marker of molt). Rutin disrupted that synchrony. I discuss how patterns of host plant resistance may be altered with a decrease, in amplitude of diurnal temperatures (as has been documented recently for temperate regions) through the uncoupling of herbivore performance and allelochemical concentration. I conclude that simultaneous consideration of fluctuating temperatures and allelochemicals is advisable when assessing the effects of temperature and allelochemicals on performance of insect herbivores because interactive effects between temperature and dietary components occur and perhaps are common.     

Prey consumption by phytoseiid spider mite predators as affected by diurnal temperature variations

The consumption rate of an ectothermic predator is highly temperature-dependent and is a key driver of pest-predator population interactions. Not only average daily temperature, but also diurnal temperature variations may affect prey consumption and life history traits of ectotherms. In the present study, we evaluated the impact of temperature alternations on body size, predation capacity and oviposition rate of the predatory mites Phytoseiulus persimilis Athias-Henriot and Neoseiulus californicus McGregor (Acari: Phytoseiidae) when presented with eggs of their natural prey, the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae). For both predators, mean daily temperature as well as temperature alternation had a substantial impact on the number of prey consumed. At lower average temperatures, more eggs were killed under an alternating temperature regime (20 °C/5 °C and 25 °C/10 °C) than at the corresponding mean constant temperatures (15 and 20 °C). At higher average temperatures (>25 °C), however, the opposite was observed with higher numbers of prey killed at constant temperatures than at alternating temperatures. At 25 °C, temperature variation had no effect on the predation capacity. A similar trend as for the predation rates was observed for the oviposition rates of the phytoseiids. Body size of N. californicus was affected both by average daily temperature and temperature variation, with smaller adult females emerging at alternating temperatures than at constant temperatures, whereas for P. persimilis, temperature variation had no impact on its body size. Our results demonstrate that temperature variations are likely to affect biological control of T. urticae by the studied phytoseiid predators.     

Diapause induction in the oblique-banded leafroller Choristoneura rosaceana (Lepidoptera: Tortricidae): Role of photoperiod and temperature

Induction of diapause in the larval stage of the oblique-banded leafroller, Choristoneura rosaceana (Harris), was found to be dependent on both photoperiod and temperature. At constant temperatures of 24, 20 and 16°C, short photoperiods induced diapause. The critical photoperiod was between 14–15 h of light per day at 20 and 16°C. At 14 h light: 10 h dark, all larvae expressed diapause. Temperature had a modifying effect, and slightly shifted the larval response to diapause-inducing photoperiods. High constant temperatures of 28°C and above induced diapause in some individuals (< 20%), while fluctuating temperatures of 32 and 16°C in a 12-h cycle resulted in 67% diapause induction, suggesting that diapause could also be induced by fluctuating temperatures, particularly if the higher temperature exceeds 25°C.The first- and the second-instar larvae were the only two stages sensitive to diapause induction. Exposure of adult, egg and third, fourth, and fifth-larval instars to diapause-inducing conditions did not produce diapause. Although diapause was induced in the first or the second instars, it was always expressed in the third or fourth instar.     

The rate of preimaginal development of the blowfly, Calliphora vicina R.-D. (Diptera, Calliphoridae) under field and laboratory conditions

Open field experiments with the blowfly, Calliphora vicina originating from the environs of St. Petersburg showed that the correlation between the rate of its preimaginal development under the natural conditions and the mean daily temperature could be very closely approximated by linear regression. The sum of effective temperatures required for development from the egg to the puparium constituted ca 140 degree-days and from the egg to the adult, ca 320 degree-days with the lower thresholds of 5.8 and 4.8°C, respectively. The minimum duration of development (6–8 days from the egg to the puparium and 16–20 days from the egg to the adult) was recorded during the period of the highest mean temperatures (22–23°C) from July 8 to August 15 of 2010 and 2011. The rate of development under natural thermorhythms was not siggnificantly different from that under the laboratory conditions at constant temperatures from 12 to 23°C.     

Temperature effects on zoeal morphometric traits and intraspecific variability in the hairy crab Cancer setosus across latitude

Phenotypic plasticity is an important but often ignored ability that enables organisms, within species-specific physiological limits, to respond to gradual or sudden extrinsic changes in their environment. In the marine realm, the early ontogeny of decapod crustaceans is among the best known examples to demonstrate a temperature-dependent phenotypic response. Here, we present morphometric results of larvae of the hairy crab Cancer setosus, the embryonic development of which took place at different temperatures at two different sites (Antofagasta, 23°45′ S; Puerto Montt, 41°44′ S) along the Chilean Coast. Zoea I larvae from Puerto Montt were significantly larger than those from Antofagasta, when considering embryonic development at the same temperature. Larvae from Puerto Montt reared at 12 and 16°C did not differ morphometrically, but sizes of larvae from Antofagasta kept at 16 and 20°C did, being larger at the colder temperature. Zoea II larvae reared in Antofagasta at three temperatures (16, 20, and 24°C) showed the same pattern, with larger larvae at colder temperatures. Furthermore, larvae reared at 24°C, showed deformations, suggesting that 24°C, which coincides with temperatures found during strong EL Niño events, is indicative of the upper larval thermal tolerance limit. C. setosus is exposed to a wide temperature range across its distribution range of about 40° of latitude. Phenotypic plasticity in larval offspring does furthermore enable this species to locally respond to the inter-decadal warming induced by El Niño. Morphological plasticity in this species does support previously reported energetic trade-offs with temperature throughout early ontogeny of this species, indicating that plasticity may be a key to a species’ success to occupy a wide distribution range and/or to thrive under highly variable habitat conditions.     

Seed germination ecology of Viola calaminaria,an endangered metallophyte with a narrow distribution

Viola calaminaria is an endangered metallophyte endemic to a small area close to the border between Belgium, Germany and the Netherlands, where it grows on rock outcrops rich in heavy metals (zinc, lead and cadmium). Because V. calaminaria reproduces mainly by seeds, it is of crucial importance to understand its germination requirements. Germination percentage and speed at constant (11–25°C) and alternating (23/09°C) temperatures were investigated in five large populations. Germination percentage was positively correlated to seed weight. Germination was low (<25%) at 11 and 16°C, intermediate (around 65%) between 20 and 25°C and the highest (93%) at the alternating temperature regime (23/09°C). V. calaminaria is a slow germinator requiring 41 days on average to germinate at 23/09°C and considerably more at 20 to 25°C (105 days on average). Our results also highlighted that the species is desiccation tolerant and can therefore be safely conserved under standard seed bank conditions.     

Thermosensitive genetic dwarfs of apple

The role of environment on the dwarfing (short internode) phenomenon of apple (Malus domestisca Borkh.) was investi gated and defined in controlled environmental chambers. Orchard-grown very dwarf, dwarf and semi-dwarf trees obtained by natural sibcrossing of spur-type cv. Golden Delicious and cv. Delicious, as well as standard cv. Golden Delicious, were propagated via in vitro techniques. Growth was rapid and none of the 4 types exhibited dwarf-like characteristics when grown at constant 27°C with 12, 14 or 16 h daylengths. Standard and very dwarf plants grew at nearly the same rate at constant 30°C, whereas growth nearly ceased on both types at constant 35°C after 7 days. Dwarf and very dwarf plants responded differently from standard and semi-dwarf plants when grown under alternating (ramped) night/day temperatures (15 or 20°C night ramped up to a daytime maximum over 8 h of 23, 28, 33 or 38°C, held for 2 h and then ramped down over 5 h to the night temperature). As the night/maximum day temperature differentia) increased from 0 to 23° under the ramping environments, growth of dwarf plants decreased markedly as compared to standard plants. When the same night/maximum day temperature differential occurred, the effect on decreasing shoot length was greater at the higher (20°C) night temperature. Increasing maximum day temperatures under the ramped environment also reduced leaf area plant^?1 but did not markedly affect leaf number, resulting in short internodes. When a period of constant temperature was followed by ramped temperatures or vice versa, the sequence of constant vs ramped environments made little difference in the final growth of the 4 plant types. The data point to high temperature as the major factor for causing dwarfing of the sensitive plant types. Increasing the differential between night and maximum day temperature resulted in short internode. dwarf plants with small leaves similar to orchard-grown dwarf trees.     

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.     

Thermal requirements for growth,survival and aerobic performance of weatherfish larvae Misgurnus fossilis

Thermal requirements of larval weatherfish Misgurnus fossilis were investigated in terms of growth, survival and aerobic performance. Growth and survival of M. fossilis larvae acclimated to five temperatures (11, 15, 19, 23 and 27° C) were measured over 25 days. In the upper temperature treatments (19, 23 and 27° C), survival of larvae was stable throughout the entire rearing period (>75%), whereas 11 and 15° C resulted in severe declines in survival (to <10%). Growth of larvae (expressed as dry mass and total length) was highest at 19 and 23° C, but significantly decreased at 27° C. Routine metabolic rate of 3 days post‐hatch larvae was estimated as oxygen consumption rate (?O₂) during acute exposure (30 min to 1 h) to seven temperatures (11, 15, 19, 23, 27, 31 and 35° C). Larval oxygen uptake increased with each consecutive temperature step from 11 to 27° C, until a plateau was reached at temperatures >27° C. All larvae of the 35° C regime, however, died within the ?O₂ measurement period. M. fossilis larvae show greater than expected tolerance of high temperatures. On the other hand, low temperatures that are within the range of likely habitat conditions are critical because they might lead to high mortality rates when larvae are exposed over periods >10 days. These findings help to improve rearing conditions and to identify suitable waters for stocking and thus support the management of re‐introduction activities for endangered M. fossilis.