Strain- and Genotype-Specific Differences in Virulence of Paenibacillus larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees

Virulence variations of Paenibacillus larvae subsp. larvae, the causative agent of American foulbrood disease of honeybees, were investigated by analysis of 16 field isolates of this pathogen, belonging to three previously characterized genotypes, as well as the type strain (ATCC 9545) of P. larvae subsp. larvae, with exposure bioassays. We demonstrated that the strain-specific 50% lethal concentrations varied within an order of magnitude and that differences in amount of time for the pathogen to kill 100% of the infected hosts (LT₁₀₀) correlated with genotype. One genotype killed rather quickly, with a mean LT₁₀₀ of 7.8 ± 1.7 days postinfection, while the other genotypes acted more slowly, with mean LT₁₀₀s of 11.2 ± 0.8 and 11.6 ± 0.6 days postinfection.     

Reduced activity of juvenile hormone esterase in microsporidia-infected Lymantria dispar larvae

Infection of the fat body of Lymantria dispar (Lep.: Lymantriinae) larvae with the microsporidium Vairimorpha disparis has severe effects on juvenile hormone (JH) metabolism of the host. Beginning 8 days postinfection, activity of the JH degrading enzyme JH-esterase was significantly lower in the hemolymph of infected than uninfected larvae. Activity remained low as microsporidiosis progressed. JH titers were slightly elevated in infected larvae; the difference was not significant in most cases. This disturbance of JH metabolism may be due to generally impaired fat body functions and high demand for resources by the developing pathogen.     

Effect of high and low temperatures on the drugstore beetle (Coleoptera: Anobiidae)

The drugstore beetle, Stegobium paniceum (L.) (Coleoptera: Anobiidae), is a pest of stored medicinal and aromatic plants. Generally, mortality of each stage increased with an increase of temperature and exposure time. Heat tolerance for different stages from highest to lowest was young larvae, old larvae, eggs, adult, and pupae. The mortality after 7 h at 42 degrees C for young larvae, old larvae, eggs, adults, and pupae, respectively, was 16 +/- 5, 31 +/- 6, 48 +/- 3, 63 +/- 8, and 86 +/- 2% (mean +/- SEM). Similar trends for stage specific mortality were seen with the lethal time for 90% mortality (LT90) at 42 degrees C; 773, 144, 12, and 11 h for old larvae, eggs, adults, and pupa respectively. Mortality was too low with young larvae to estimate LT90. The LT90 for young larvae at 42, 45, 50, 55, and 60 degrees C was 25, 20, 3.9, 0.18, and 0.08 h, respectively. The cold tolerance of different stages at 0 degree C from highest to lowest was adults, old larvae, young larvae, pupae, and eggs. The LT90 at 0 degrees C was 298, 153, 151, 89, and 53 h, respectively. The LT90 for adults at 5, -5, -10, and -15 degrees C was 792, 58, 2, and 0.8 h, respectively. The supercooling point of adults was -15.2 +/- 2 degrees C; young larvae, -9.0 +/- 0.8 degrees C; old larvae, -6.5 +/- 0.5 degrees C; and pupae, -4.0 +/- 1.4 degrees C. Heat treatments that control young larvae should control all other stages of S. paniceum. Cold treatments that control adults should control all other stages of S. paniceum. Dried plants stored at 5 degrees C for 45 d or 42 degrees C for 30 h and then kept below 18 degrees C throughout the rest of the year, should remain pest-free without any chemical control.     

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology,structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.     

Trichinella spiralis: specificity of ES antigens from pre-encysted larvae.

Excretory/secretory (ES) antigens were obtained by culturing pre-encysted Trichinella spiralis larvae which were recovered from muscles of experimentally infected mice 14-15 days postinfection. Analyses of these antigens (PEL ES) with immunoblotting, SDS-PAGE and Triple Antibody ELISA showed that they yielded a low sensitivity and specificity when tested with antisera against the common nematodes of Chinese pigs. As compared to ES antigens from encysted larvae, PEL ES also contained more low molecular mass proteins.     

Pseudomonocystis sp., an Eugregarine Protozoan Pathogen of the Coconut Root Grub, Leucopholis coneophora

Populations of the coconut root grub , Leucopholis coneophora, were infected by an eugre garine protozoan pathogen , Pseudomonocystis sp . The pathogen infected 22.7% third - instar L. coneophora larvae in the field . The infected larvae produced 1 - 84 (average 37 . 11) milky - white , membranous , elliptical cysts measuring 2 . 64 1 . 24 mm . Each cyst contained about 3 . 80 105 mucilaginous spores . The spores were elliptical with two tubular polar caps , thick - walled , ornamented with irregular ridges and furrows , and measured 26 . 04 13 . 61 mum . The LD and LT for oral inoculation of spores into third - instar larvae were 9 . 86 104 50 50 spores / larva and 27 . 62 days respectively . Ingestion of spores with or without food and connibalism of infected larvae were the modes of transmission of the pathogen .     

Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10^–2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10^–3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.     

Plasma FSH and LH in prepubertal Booroola ewe lambs

Basal plasma concentrations (four 30-min samples) and GnRH-induced release of gonadotrophins were measured every 15 days between 30 and 90 days and at 110 days of age in Merino ewe lambs from the prolific Booroola ('B') flock (n = 18-23), the medium prolificacy ('T') flock (n = 14-20), and the 'O' flock (n = 4-8) of low prolificacy. At ages of 30 and 45 days B ewe lambs had mean basal plasma FSH concentrations of 145 and 122 ng/ml which were significantly higher (P less than 0.01) than those seen in T (45 and 53 ng/ml), and O (39 and 38 ng/ml) flock ewes. Between 60 and 110 days of age there were no significant differences between genotypes. The increment in FSH concentrations above basal levels induced by the subcutaneous injection of 100 micrograms synthetic GnRH was only significantly (P less than 0.05) greater in B than T and O genotype ewe lambs at 110 days of age but not at other ages. The basal plasma FSH differences between the B, T and O genotypes at 30 and 45 days of age were not consistently related to the size of litter in which lambs were born. At 30 days of age the mean plasma LH concentration of B, T, and O flock lambs were 2.6 +/- 0.5, 1.2 +/- 0.6 and 0.7 +/- 0.8 ng/ml respectively. These differences were not significant. At later ages there were also no significant differences between the genotypes with respect to basal LH, and the increase in LH induced by exogenous GnRH was always similar for the three genotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antixenosis in maize reduces feeding by western corn rootworm larvae (Coleoptera: Chrysomelidae)

SUM2162 is the first known example of a naturally occurring maize, Zea mays L., genotype with antixenosis (nonpreference) resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding. Behavioral responses of neonate western corn rootworm larvae were evaluated in laboratory bioassays with seven maize genotypes selected for native resistance to rootworm feeding damage. Two susceptible maize genotypes and one transgenic (Bacillus thuringiensis) maize genotype were included as controls. In soil bioassays with cut roots, no larvae entered the roots of the resistant variety SUM2162, but at least 75% of the larvae entered the roots of every other maize type. Larvae made significantly fewer feeding holes in the roots of SUM2162 than in all the other maize genotypes, except the isoline control. In feeding bioassays, larval feeding varied significantly among maize genotypes, but there was no significant difference between the resistant varieties and the susceptible controls. There were no significant differences among any of the genotypes in host recognition (search) behavior of larvae after exposure to the roots. Little variation in feeding stimulant blends was observed among maize genotypes, indicating minimal contribution to the observed antixenosis.     

Epizootiology of Eustrongylides ignotus in Florida: transmission and development of larvae in intermediate hosts

Under laboratory conditions, 2 modes of transmission of Eustrongylides ignotus (Nematoda: Dioctophymatoidea) to fish were identified. Eastern mosquitofish (Gambusia holbrooki) became infected after ingestion of either eggs of E. ignotus containing first-stage larvae or aquatic oligochaetes (Limnodrilus hoffmeisteri) containing third-stage larvae of E. ignotus. After removal from the uterus of gravid E. ignotus females and incubation for 17-28 days, depending on temperature, it was found that parasite eggs contained first-stage larvae that were infective to fish and oligochaetes. Larvae developed to the third stage in oligochaetes and were infective to fish 35-77 days postinfection (PI) and when fed to fish, developed to the fourth stage between 127 and 184 days PI. Eggs containing first-stage larvae fed directly to fish developed to the fourth stage between 84 and 105 days PI. The amount of time for development from the undifferentiated egg to the fourth-stage larva was 78-156 days shorter when fish ingested eggs containing first-stage larvae than when fish ingested oligochaetes containing third-stage larvae. Three species of large piscivorous fish, including black crappie (Pomoxis nigromaculatus), largemouth bass (Micropterus salmoides), and warmouth (Lepomis gulosus), were fed mosquitofish containing fourth-stage larvae. At necropsy, live E. ignotus larvae were recovered from all 3 species. Several fish had multiple infections after ingesting > 1 larva, indicating that bioaccumulation of the parasite in the food chain may occur.     

In vitro rearing Oestrus caucasicus third-instar larvae and pupae (Diptera: Oestridae) from naturally-infested Iberian ibex, Capra Pyrenaica (Artiodactyla: Bovidae)

Third-instar Oestrus caucasicus larvae (n = 236) obtained from Iberian ibex, Copra pyrenaica, were reared in a laboratory to obtain adult flies. They were maintained at a temperature of 21.9 +/- 2.7 degrees C and a relative humidity of 38.9 +/- 8.0 %. In all, 78 imagos emerged (33.1 %), with a sex-ratio at emergence not differing significantly from 1:1; 25 larvae did not complete pupariation. A total of 14 adult flies (17.9 % of the adults obtained) showed malformations, mainly in their wings. The pupariation period lasted around 30 hours and the pupal stage lasted on average 29.8 +/- 6.8 days. The success of pupation in both sexes was mainly determined by the weight of the larvae. Sexual dimorphism, with higher weights in females, was evident in third-instar arvae, pupae and adults. The mean longevity of adult flies was 224.8 +/- 91.4 hours and males generally survived for onger than the females.     

Control of diapause in codling moth larvae

Diapause in a New Zealand strain of codling moth (Cydia pomonella Linnaeus [Lepidoptera: Olethreutidae]) was induced in larvae by photoperiods of 15 h or less. Once diapause had been initiated, it could not be terminated by any combination of conditions tested for at least 20 days after cocooning. In diapausing larvae a low rate of pupation occurred at 25 °C under a long day (18 h) photoperiod. A high rate of pupation was achieved under a long day regime when larvae were decocooned, and provided with apple as nourishment. Diapause could be terminated predictably in 94–100% of larvae by 1) conditioning at 15 °C and constant darkness for periods of 40–100 days, then 2) chilling at 2±2 °C and constant darkness for 20–50 days followed by 3) any post-chill condition periods at 25 °C, 18 h photoperiod. Complete diapause termination was achieved when 100 days conditioning was followed by 30 days or 50 days post-chill period. Under these conditions, 76% termination occurred in the post-chill period after 10 days, and 93% after 25 days.To terminate diapause in codling moth larvae, we recommend that a 100 days conditioning followed by 30 days chilling and 50 days post chilling periods be used.     

Development of third-stage Physaloptera larvae from Crotalus viridis rafinesque, 1818 in cats with notes on pathology of the larvae in the reptile. (Nematoda, Spiruroidea)

The prairie rattlesnake, Crotalus viridis, was found to be commonly infected with third-stage Physaloptera larvae. A total of 112 larvae were fed to 3 laboratory raised cats. Adult worms recovered 42 and 59 days postinfection were identified as P. rara. Observations were made on the pathology of the larvae in the snake.     

Differentiation of Paenibacillus larvae subsp. larvae,the cause of American foulbrood of honeybees,by using PCR and restriction fragment analysis of genes encoding 16S rRNA

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.     

Food consumption by Chilo partellus (Lepidoptera: Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding natural versus artificial diets on mortality and mycosis

Second and third instar Chilo partellus larvae were infected with Beauveria bassiana and Metarhizium anisopliae (both at 1x10(8)conidia/ml) and daily consumption of maize leaves was measured. Infection by the fungi was associated with reduced mean daily food consumption. Reduction in food consumption became evident 3-4 days after treatment with the fungi for second instar larvae and 4-5 days for third instar larvae. Four conidial concentrations, 1x10(5), 1x10(6), 1x10(7), and 1x10(8)conidia/ml, were tested against second instar larvae. Food consumption dropped by 70-85% when the second instar larvae were treated with the fungi at 1x10(8)conidia/ml. Reduction in food consumption by C. partellus larvae infected with B. bassiana and M. anisopliae may offset the slow speed of kill of the fungi. The effect of artificial versus natural diets on mortality and mycoses of second instar larvae treated with the fungi at 1x10(8)conidia/ml was determined. Larvae provided with artificial diet suffered little mortality and mycoses than larvae provided with maize leaves. The LT(50) was longer for larvae provided with artificial diet.     

Evaluation of Metarhizium anisopliae (Metsch) Sorok. to target larvae and adults of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) in soil and fiber band applications

The aim of this work has been to evaluate in the laboratory the potential of entomopathogenic fungi against adults and larvae of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) through fiber band application and a potted plant bioassay with soil application, respectively. Our previous findings revealed that Metarhizium anisopliae EAMa 01/58-Su isolate was the most virulent against neonate larvae of the buprestid. In the present work, M. anisopliae EAMa 01/58-Su isolate has been also shown to be highly virulent against adult beetles by immersion in a conidial suspension; thus it was selected to accomplish our objectives. When adult beetles were stimulated to climb 100 x 200 mm non-woven commercial fiber bands impregnated with conidia of M. anisopliae EAMa 01/58-Su isolate, total mortality rates varied from 85.7% to 100.0%; whereas no significant correlation was detected between the time needed to cross the band (mean value 648.7+/-22.4s) and the time of death, with mean average survival time ranging between 10.3 and 16.0 days, compared to 28 days of the controls. Potted seedlings (5-6 months old) of cherry plum (Prunus myrobalana Lois.), a commonly used apricot rootstock, were used to study the efficacy of soil treatment with M. anisopliae EAMa 01/58-Su isolate against neonate C. tenebrionis larvae. The soil inoculation with M. anisopliae EAMa 01/58-Su isolate had a significant effect on the mean number of dead larvae recovered from the roots, with mean mortality ranging from 83.3% to 91.6%; whereas no significant differences were detected between the three fungal doses. In all cases, dead larvae found within roots exhibited external signs of fungal growth. Hence, it may be possible to use M. anisopliae EAMa 01/58-Su isolate in a biocontrol strategy targeting both adults and larvae of C. tenebrionis.     

Preventing ascidian fouling in aquaculture: screening selected allelochemicals for anti-metamorphic properties in ascidian larvae

Fouling by ascidians causes major stock losses and disrupts production in marine aquaculture, especially bivalve aquaculture. Currently, no cost effective solution exists despite the testing of many prospective control techniques. This study examined a range of allelochemicals suspected to inhibit metamorphosis in marine larvae. Five allelochemicals were screened in a larval metamorphosis bioassay using Ciona savignyi Herdman to determine their potential as a remedy for ascidian fouling in bivalve aquaculture. Three of the compounds tested inhibited ascidian larval metamorphosis and increased mortality at low concentrations. These were radicicol (99% inhibition of metamorphosis [IC??], 0.8 μg ml?1; 99% lethal concentration [LC??], 2.5 μg ml?1; 99% lethal time [LT??], 7.0 days), polygodial (IC??, 0.003 μg ml?1; LC??, 0.9 μg ml?1; LT??, 6.4 days), and ubiquinone-10 (IC??, 3.2 μg cm?2; LC??, 14.5 μg cm?2; LT??, 5.6 days; expressed as μg cm?2 due to insolubility in water and ethanol). While spermidine significantly affected metamorphosis and mortality of C. savignyi, the effect was insufficient to achieve inhibition in 99% of larvae over the 7-day timeframe of the assay. Muscimol did not affect metamorphosis or mortality at the concentrations tested. The present study demonstrates that radicicol, polygodial and ubiquinone-10 have potential for future development in antifoulant formulations targeted towards the inhibition of metamorphosis in ascidian larvae, while spermidine and muscimol appear unsuitable.     

Method for diagnosis and control of aerobic training in rats based on lactate threshold

We propose a protocol for determination of lactate threshold (LT) and test the validity of one aerobic training based on LT in rats. In group I, V(LTi) (velocity at LT before training) was determined in all rats (n=10), each rat training at its own V(LTi) and in group II, animals (n=7) ran at 15 m min(-1), the mean V(LTi) of group I. The training consisted of daily runs at V(LTi) for 50 min, 5 days/week, for 4 weeks. In group I, this program increased V(LT) (V(LTi) 14.90+/-1.49 m min(-1) and V(LTf), after training, 22.60+/-1.17 m min(-1)) and the velocity at exhaustion (19.50+/-1.63 m min(-1) and 27.60+/-1.17 m min(-1)). [Lactate] at LT (2.62+/-0.43 mmol L(-1) versus 2.11+/-0.15 mmol L(-1)) and relative values of LT (76+/-3% versus 82+/-2%) stayed unaltered. In group II the V(LTf) was 20+/-1.8 m.mim(-1), the [lactate] at the LT, 2.02+/-0.17 mmol.L(-1); the exhaustion speed, 23.57+/-2.11 m.mim(-1) and relative value of LT, 82.71+/-2.29%. There were no significant differences in these parameters between groups I and II. Thus, this protocol based on LT is effective and the mean V(LT) determined in a small number of healthy untrained rats can be used for aerobic training in a larger group of healthy animals of same gender and age.     

Effect of power, pedal rate, and force on average muscle fiber conduction velocity during cycling.

Muscle fiber conduction velocity (MFCV) provides indications on motor unit recruitment strategies due to the relation between conduction velocity and fiber diameter. The aim of this study was to investigate MFCV of thigh muscles during cycling at varying power outputs, pedal rates, and external forces. Twelve healthy male participants aged between 19 and 30 yr cycled on an electronically braked ergometer at 45, 60, 90, and 120 rpm. For each pedal rate, subjects performed two exercise intensities, one at an external power output corresponding to the previously determined lactate threshold (100% LT) and the other at half of this power output (50% LT). Surface electromyogram signals were detected during cycling from vastus lateralis and medialis muscles with linear adhesive arrays of eight electrodes. In both muscles, MFCV was higher at 100% LT compared with 50% LT for all average pedal rates except 120 rpm (mean +/- SE, 4.98 +/- 0.19 vs. 4.49 +/- 0.18 m/s; P < 0.001). In all conditions, MFVC increased with increasing instantaneous knee angular speed (from 4.14 +/- 0.16 to 5.08 +/- 0.13 m/s in the range of instantaneous angular speeds investigated; P < 0.001). When MFCV was compared at the same external force production (i.e., 90 rpm/100% LT vs. 45 rpm/50% LT, and 120 rpm/100% LT vs. 60 rpm/50% LT), MFCV was higher at the faster pedal rate (5.02 +/- 0.17 vs. 4.64 +/- 0.12 m/s, and 4.92 +/- 0.19 vs. 4.49 +/- 0.11 m/s, respectively; P < 0.05) due to the increase in inertial power required to accelerate the limbs. It was concluded that, during repetitive dynamic movements, MFCV increases with the external force developed, instantaneous knee angular speed, and average pedal rate, indicating progressive recruitment of large, high conduction velocity motor units with increasing muscle force.     

Effect of exposure method to Beauveria bassiana and conidia concentration on mortality, mycosis, and sporulation in cadavers of Chilo partellus (Lepidoptera: Pyralidae)

The effects of exposure methods, conidial concentrations, and temperature on mortality, mycosis and sporulation in second instar Chilo partellus cadavers infected by Beauveria bassiana was investigated in laboratory studies. Larvae directly sprayed with conidia, exposed to conidia-treated leaves, and dipped into conidial suspension resulted in high mortality (98-100%). The longest LT50 (3.5 days) and days to mortality (2.6 days) were observed in the treated-leaves exposure method. The shortest LT50 (1 day) and days to mortality (1 day) were recorded for the dipping method. With increasing conidial concentrations, there were decreasing LT50 and days to mortality. Larvae exposed to treated leaves and larvae directly sprayed with conidial suspensions produced high mycoses in cadavers. Exposure of larvae to treated-leaves resulted in high sporulation. At lower concentrations of conidia, both mycosis and sporulation in cadavers were high. The optimum temperature for mycosis was 20 and 15 degrees C for sporulation.