Adult honeybee's resistance against Paenibacillus larvae larvae, the causative agent of the American foulbrood.

American foulbrood is a widespread disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae subsp. larvae. Spores represent the infectious stage; when ingested by a larva they germinate in the midgut. The rod-shaped vegetative forms penetrate the larva's intestinal tissue and start multiplying rapidly, which finally kills the larva. Spores fed to adult honeybees, however, do not harm the bees. We investigated this phenomenon. Specifically, we studied the influence of the adult honeybee midgut on the vegetative growth and on the germination of spores of P. larvae larvae. We focused on two groups of adult workers that are likely to have large numbers of spores in their gastrointestinal tracts in infected colonies: middle-aged bees, which are known to remove or cannibalize dead larvae and clean brood cells, and winterbees, which do not have frequent chances to defecate. We found that midgut extract from winterbees and worker-aged bees of different colonies almost completely inhibited the growth of the vegetative stage of P. larvae larvae and suppressed the germination of spores. The inhibiting substance or substances from the adult midgut are very temperature stable: they still show about 60% of their growth-inhibiting capacity against this bacterium after 15 min at 125 degrees C. We established a method to test growth-inhibiting factors against P. larvae larvae in vitro.     

Regional distribution of Paenibacillus larvae subspecies larvae, the causative organism of American foulbrood, in honey bee colonies of the Western United States

We examined honey bee, Apis mellifera L., colonies pollinating almonds in California during February 2003 for Paenibacillus larvae subsp. Larvae, the causative organism of the virulent brood disease American foulbrood. Colonies originating from the Rocky Mountain area and California had significantly higher numbers (P < 0.05) of bacterial colony-forming units (CFUs) (408 and 324 per 30 adult bees, respectively) than colonies from the upper Midwest (1.28). Colonies from the northwestern, central, and southwestern United States had intermediate CFU or bacterial colony levels. Operations positive for P. larvae larvae were relatively uniform at approximately 70-80%, and no regional significant differences were found. Percentages of colonies with high CFUs (> or = 400 per 30 bees) differed significantly, with those from the Rocky Mountain region having 8.73% compared with those of the upper Midwest with 0%. The significance of CFU levels was evaluated by inoculating healthy colonies with diseased immatures and sampling adult bees. The number of CFUs detected per diseased immature was conservatively estimated to be approximately 399 CFUs per 30 adult bees. We defined this spore level as 1 disease equivalent. Based on this, 3.86% colonies in our survey had 1 or more disease equivalent number of P. larvae larvae CFUs. Operations with high P. larvae larvae spore levels in their colonies will likely observe American foulbrood if prophylaxis is not practiced diligently.     

Daily changes in the sensitivity of wax-moth larvae, Galleria mellonella, to cooling stress

ABSTRACT. Sensitivity to cooling stress in the last instar larvae of Galleria mellonella was measured as (a) the number of extra larval moults, (b) the number of larvae retaining the ability to secrete silk, and (c) the number showing arrested development. With respect to (a) and (b) there were considerable differences in sensitivity across the day. A relationship was observed between the number of additional larval moults induced by chilling and the ability of prepupal larvae to spin silk: the periods during the 24 h when the most larvae passed through additional larval moults were periods characterized by the smallest number of larvae capable of spinning, and vice versa. These daily changes were apparently partly independent of developmental age. Daily variations in sensitivity also occurred when larvae of the same age were cooled at different times of day. It is suggested that these rhythms in cold-sensitivity are related to a cold-sensitive rhythm in juvenile hormone secretion, or hormone sensitivity in the tissues.     

Pathogenicity of fungi isolated from the larvae of the mangrove crab,Scylla serrata, in Indonesia

Heavy mortality reaching almost 100% occurred in the larvae of the mangrove crab,Scylla serrata, from July to December 1997 at the hatchery of Gondol Research Station for Coastal Fisheries in Bali, Indonesia. Mortality was observed in larvae after hatching from the eggs. The affected larvae were whitish and filled with numerous aseptate hyphae. Three fungi belonging to the order Lagenidiales,Lagenidium callinectes, Haliphthoros milfordensis, andHalocrusticida sp., were isolated from the infected larvae. Pathogenicity tests of the infected fungi against the larvae of mangrove crab demonstrated that all isolates were pathogenic.     

Recycling of urea associated with the host plant urease in the silkworm larvae,Bombyx mori

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.     

Salinity tolerance of laboratory-reared larvae of the California killifish, Fundulus parvipinnis Girard

Newly hatched larvae of the California killifish ( Fundulus parvipinnis ) reared in the laboratory, were tolerant of salinities from fresh water to 70‰. Their salinity tolerance was influenced by incubation salinity; larvae hatched in lower incubation salinities exhibited greater freshwater tolerance than those hatched in higher salinities. In gradual acclimation tests, the upper median lethal salinity for the larvae was 130‰. Freshwater tolerance of the larvae decreased with age; yolk sac larvae were completely tolerant of fresh water while larvae more than 15 days old were least resistant.     

Ecdysteroid production during caste differentiation in larvae of the ant, Plagiolepis pygmaea

ABSTRACT. Variations in ecdysteroids were measured by radio-immunoassay in worker-biased and queen-biased larvae of the ant Plagiolepis pygmaea Latr. (Hymenoptera, Formicidae) during late larval development, i.e. from the end of winter diapause up to the prepupal period. At the end of diapause, larvae are bipotential and, depending on culture conditions, can become either queens or workers. Ecdysteroid profiles revealed that there are striking differences between the two castes: worker larvae showed high titres during their development, queen larvae had low titres over the same period.     

Cold hardiness of larvae of the southwestern corn borer, Diatraea grandiosella

The cold-hardening capacity of field-collected larvae from southeast Missouri and laboratory-reared larvae of the southwestern corn borer, Diatraea grandiosella Dyar, was examined. Supercooling points of non-diapause and diapause larvae collected from maize plants grown in Missouri (36°30 N lat.) were ca.-7.0°C. The hemolymph melting points of diapause field larvae (-0.8°C) were significantly lower than those of non-diapause larvae collected in July (-0.5°C). The supercooling points of hemolymph from non-diapause and diapause field larvae ranged randomly from-10° to-18°C. Supercooling points of non-diapause laboratory larvae increased from-13° to-10°C prior to pupation, whereas those of diapause larvae increased similarly before the onset of diapause, but then decreased during diapause to ca.-17°C. No change in supercooling points or capacity to survive in the presence of ice was observed in diapause laboratory larvae acclimated at 4°C for 63 days. Laboratory and field larvae began to freeze at ca.-1.5°C in the presence of ice, but survived to several degrees below their melting points. The high supercooling points of field larvae appeared to be due to the presence of an environmental ice-nucleator. Although data for laboratory larvae indicate sufficiently low supercooling points to permit winter survival in southeastern Missouri, considerable larval mortality occurs in the field due to inoculative freezing and the presence of an ice-nucleator.     

Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.     

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.     

Neural correlates of settlement in veliger larvae of the gastropod,Crepidula fornicata

Settlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10^?5 mol L^?1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.     

Dispersal strategies in sponge larvae: integrating the life history of larvae and the hydrologic component

While known to be uniformly non-feeding, short-lived, and potentially short dispersing, sponge larvae display different behaviours (swimming ability and taxis). Our aim was to show whether sponge larvae with different behaviours exhibit different dispersal strategies under variable intensity of water movements. We first assessed the distribution of larvae of six taxa: Dictyoceratida spp., Dysidea avara, Crambe crambe, Phorbas tenacior, Scopalina lophyropoda, and Cliona viridis, collected through plankton sampling, and the abundance of the corresponding adult sponges across three hard bottom communities and a sandy bottom from a north-west Mediterranean rocky shore. We then tested adult–larvae couplings (abundance of larvae vs abundance of adults) under increasing levels of water movements (surge) to assess the importance of this environmental factor in driving differences in dispersal strategies. Adults of Dictyoceratida spp., D. avara, and P. tenacior were most abundant in semi-dark caves (SDC), C. crambe and C. viridis in communities of sciaphilic algae (SA), whereas the distribution of S. lophyropoda was extremely patchy, being present almost only in the SA community of one of the five stations studied. Larvae of Dictyoceratida spp. and P. tenacior were more abundant in the SDC, whereas D. avara and C. crambe were homogeneously distributed across the communities. The larvae of C. viridis were more abundant in the SA communities and the S. lophyropoda larvae were mostly present in one station and one community (SA). Increased water movement did not modify the adult–larvae coupling for Dictyoceratida spp., D. avara, and C. crambe, whereas it broke up the positive association for P. tenacior and to some extent S. lophyropoda. For C. viridis, possible variability in adult–larvae coupling was not tested because the larvae were collected on only one day under calm sea conditions. We confirm that efficient-swimming larvae with some cue response can actively counteract hydrodynamic forces and highlight the importance of both larval behaviour and environmental conditions in determining small-scale patterns of dispersal.     

Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Larvae of Culex quinquefasciatus are much more susceptible to the toxin of Bacillus sphaericus than are larvae of Aedes aegypti. In the present study, the rate of ingestion, dissolution, and the cleavage by midgut proteases of the B. sphaericus toxin were compared in larvae of these species to determine whether these factors account for the differences in susceptibility. During filter feeding, larvae of both species removed significant quantities of B. sphaericus toxin from suspensions. Filtration rates for 1 hr, the time at which C. quinquefasciatus exhibited marked intoxication, were higher for A. aegypti (576-713 microliters/larva/hr) than for C. quinquefasciatus (446-544 microliters/larva/hr). Within 24 hr of exposure, A. aegypti larvae ingested 97-99% of the toxin particulates and suffered not more than 10% mortality in suspensions which induced complete mortality in C. quinquefasciatus within 2 hr of exposure. Quantification of the particulate toxin present in larvae after exposure to B. sphaericus suspensions revealed that larvae of both species contained only minor amounts of the toxin, suggesting the larvae had been able to solubilize the toxin after ingestion. Proteases recovered from the feces of larvae cleaved at 43-kDa protein isolated from B. sphaericus toxin extract to 40 kDa in both species. Thus, differences in susceptibility to the B. sphaericus toxin between A. aegypti and C. quinquefasciatus are not due to differences in rates of ingestion, dissolution, or the specificity of proteases.     

Epibenthie fish larvae in the Great Barrier Reef Lagoon near Lizard Island,Australia

Epibenthic fish larvae near Lizard Island in the Great Barrier Reef Lagoon were sampled with a plankton sled during daylight in November 1981 and January–February 1982. Abundance in the epibenthos was highly variable, and although many types of larvae were present, few were concentrated there relative to the water column. Among those taxa concentrated in the epibenthos, abundances were low and variances were high. Larvae of bregmacerotids, callionymids, clupeids, monacanthids, pinguipedids, platycephalids, pseudochromids, and especially schindleriids, leiognathids and terapontids were concentrated in the epibenthos. Few reef fish larvae were epibenthic. There was some evidence of diel and ontogenetic movements into and out of the epibenthos. Our limited sampling indicates that conventional midwater plankton sampling is adequate for most fish larvae found in the Lizard Island area, but for the larvae of the above ten families, this could produce large underestimates of abundance.     

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.     

Chemical composition of newborn larvae, muscle larvae and adult Trichinella spiralis

Chemical composition of newborn larvae, muscle larvae and adult Trichinella spiralis. International Journal for Parasitology16: 455–460. The chemical composition of newborn larvae (NBL), muscle larvae (ML) and 4-day-old and 7-day-old adult Trichinella spiralis were compared. Total protein constituted 44.1% of the dry wt of ML, 36.9% of NBL and 30.3% of 7-day-old adult worms. Glycogen content was 16.1% of worm dry wt in ML and 7.8% in NBL and 7.2% of worm dry wt in adults. Trehalose content was 5.2% in NBL, 8.3% in ML and 8.7% in 7-day-old adult worms. Significantly lower levels of trehalose and glycogen were found in 4-day-old worms than were present in 7-day-old worms. Free glucose amounted to 0.1% of dry wt in ML, 0.37% in NBL and 0.87% in 7-day-old adults. RNA accounted for 3.5% of the dry wt of ML, 3.2% of the dry wt of 7-day-old adults and 1.1% of the dry wt of NBL. The DNA content of NBL was 0.23% of worm dry wt, in ML 0.48% of worm dry wt and 0.51% of worm dry wt in 7-day-old adults. The three parasite stages examined agreed closely with regard to types of amino acids in the free pool. Exceptions were as follows: NBL lacked taurine which both adults and ML contained; adults lacked methionine which both ML and NBL contained; and, trace amounts of cysteine were present in ML but absent from the other two stages.     

Balanomorph barnacle larvae in the plankton at McMurdo Sound,Antarctica

Summary Ten larvae of a balanomorph barnacle were collected in plankton samples from under the sea-ice of McMurdo Sound in the austral spring 1985. This is the first record of planktonic barnacle larvae in the Antarctic Ocean. By deduction they are identified as Bathylasma corolliforme (Hoek) stage II nauplii and cyprids. They are described, the first of larvae of any species of the Bathylasmatidae. Their occurrence in McMurdo Sound is discussed in view of the apparent absence of adults in the Sound.     

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.     

Proteins synthesized and secreted by larvae of the ectoparasitic wasp, Eulophus pennicornis

A microscopic examination of Eulophus pennicornis larvae on their host Lacanobia oleracea, revealed that peristaltic waves travelled from the anterior to posterior end of the feeding wasp larvae, and vice versa. In addition, when wasp larvae were immersed in PBS in vitro, they released a variety of proteins, with molecular weights ranging from (at least) 14 to 200 kDa. Amongst these was a protein with an estimated molecular weight similar to that of the 27 kDa parasitism-specific protein (PSP) detected in plasma from parasitized L. oleracea [Richards and Edwards, Insect Biochem Mol Biol 29:557-569 (1999)]. Similar results were obtained when the wasp larvae were incubated on balls of cotton wool soaked in tissue culture medium or sucrose, i.e., conditions that resemble their natural feeding behaviour. These results (and others) indicate that the wasp larvae release proteins, putatively through their mouth. Protein synthesis studies using (35)S-methionine indicated that the wasp larvae synthesize and secrete a variety of proteins in vitro, including one with a molecular weight corresponding to that of the L. oleracea 27 kDa PSP. As expected, only a portion of the total proteins synthesized by the parasitoid larvae were subsequently secreted. In addition, the autoradiogram of secreted proteins contained significantly fewer bands than silver-stained SDS gels of proteins released into PBS or onto cotton wool. Thus, some of the additional bands detected on the latter gels are thought to represent proteins that were not of wasp origin. Instead, these proteins released by the wasp larvae are speculated to be derived from their gut and, as such, probably represent proteins derived from host haemolymph and ingested during feeding. This possibility was supported by an electrophoretic analysis of homogenate supernatants prepared from wasp larvae with or without their gut contents. These studies indicated that the gut contents of the larval parasitoid contributes several distinct bands to the total protein profile. The ability of E. pennicornis larvae to synthesize, secrete, and release proteins is discussed with reference to those produced by endoparasitoid larvae. Published 2001 Wiley-Liss, Inc.