A multi-gene phylogeny provides additional insight into the relationships between several Ascosphaera species.

Ascosphaera fungi are highly associated with social and solitary bees, with some species being pathogenic to bees (causing chalkbrood) while others are not, and proper identification within this genus is important. Unfortunately, morphological characterizations can be difficult, and molecular characterizations have only used one genetic region. We evaluated multiple phylogenies of the Ascosphaera using up to six loci: the Internal Transcribed Spacer (ITS) region, 18S rRNA, 28S rRNA, Elongation Factor-1α (EF-1α) the RNA polymerase II largest subunit (RPB1), and the second largest subunit (RPB2). The ITS sequence alone produced an inadequate phylogeny, and the addition of both the 18S and 28S rRNA loci to the ITS sequence produced a phylogeny similar to that based on all six genetic regions. For all phylogenies, Ascosphaera torchioi was in a separate clade that was the most basal, with a strong genetic similarity to Eremascus albus, introducing the possibility of paraphyly within Ascosphaera. Also, based on this new phylogeny, we now suggest that the Apis mellifera (honey bee) pathogens arose within a group of saprophytes, and the Megachile (leafcutting bees) pathogens arose separately.

Relationship of Imported Fire Ant (Hymenoptera: Formicidae) Integument Coloration to Cuticular Hydrocarbon and Venom Alkaloid Profiles.

Red imported fire ant (Solenopsis invicta Buren; RIFA) and black imported fire ant (Solenopsis richteri Forel (Hymenoptera: Formicidae); BIFA) are considered distinct species with introgression via a reproductively functional hybrid (HIFA). The RIFA and BIFA common names are based on relative coloration. Due to human color perception variation, using color to identify RIFA, BIFA or HIFA is challenging. Fire ant identification traditionally involves molecular or chemical techniques, but a colorimetric test could allow rapid and low-cost identification. In this study, ant integument coloration was measured by spectrophotometer, and color attributes were compared to a combined gas chromatography (GC) index derived from cuticular hydrocarbon and venom alkaloid indices. Significant Pearson Correlation coefficients were found for colony GC index versus color attributes red to green (a*), blue to yellow (b*), chroma (C*), and hue (h*), but not lightness (L*). The RIFA colonies were distinct from BIFA for four of five color attributes and plots of the a*b* and C*h* horizontal axis of the L*a*b* and L*C*h* color spaces. Color attributes for HIFA indices were not distinct from BIFA and RIFA parental species, but HIFA a*b* and C*h* plots were distinct from RIFA and a*b* plots from BIFA. Color attributes a*, b*, and C* increased and h* decreased with GC index in a sinusoidal pattern. In conclusion, most RIFA and BIFA color attributes were distinct and a*b* and C*h* color axes plots had potential to distinguish HIFA from parental species, but coloration of HIFA indices was variable and complicated identifications among HIFA phenotypes, RIFA and BIFA.

Worker Size, Geographical Distribution, and Introgressive Hybridization of Invasive Solenopsis invicta and Solenopsis richteri (Hymenoptera: Formicidae) in Tennessee.

Worker size and geographical distribution of red imported fire ants (Solenopsis invicta Buren), black imported fire ants (Solenopsis richteri Forel), and their hybrid (S. invicta × S. richteri) (Hymenoptera: Formicidae) were evaluated from colonies sampled across Tennessee. The fire ant species and hybrid status were determined using cuticular hydrocarbon and venom alkaloid indices obtained from gas chromatography and mass spectrometry. Hybrids were the most common fire ant throughout Tennessee. With the exception of a few isolated S. invicta samples, only hybrids were found in east Tennessee, and hybrids predominated in middle Tennessee. In west Tennessee, mixed populations of S. richteri and hybrids were found. Hybrids were more common in west Tennessee than a survey performed a decade earlier. No statistical differences were detected in the average inter-colonial worker size of S. richteri and hybrids. Likewise, average worker size was not related to geographic location in Tennessee. The similarity in average worker size among hybrid colonies with a wide range of cuticular hydrocarbon and venom alkaloid values suggests introgression was not impacting ant size in colonies sampled throughout Tennessee.

The compound eye in the opaque-eye phenotype of Drosophilia melanogaster.

The compound eye of the opaque-eye mutant of Drosophila melanogaster was investigated by means of electron microscopy to determine the morphological and physical properties of ommatidial elements. These elements in the mutant were found to differ from those of the wild-type flies in the following ways: (1) The cuticular lens was thinner than that of the control and lacked the typical lamellar construction. (2) The Semper cells were irregular in shape and contained many membranous inclusions similar to those found in degenerating cells; also their nuclei contained virus-like particles. (3) The primary pigment cells contained an abundance of drosopterin-containing granules which were lacking in those of wild-type flies. (4) The superior and inferior central photoreceptor cells were misplaced and their rhabdomeres evidenced some degeneration. (5) The secondary pigment cells had only one type of pigment granules instead of the three types found in the control. These morphological changes in ommatidial elements induced physical abnormalities such as the apparent opaqueness of the eye, the lack of a pseudopupil, the probable disability of the photoreceptor cells to respond to light and the inability of the dioptric system to produce utilizable geometric images.

The fine structure of the developmental stages of the microsporidian Nosema apis Zander.

Schizonts, sporonts and sporoblasts of Nosema apis from honey bees collected in the summer and winter were studied with the electron microscope. The nuclei usually had a diplokaryon arrangement. Intranuclear spindles with polar vesicles were associated with division. Schizonts had a single limiting unit membrane, whereas sporonts had a two-layered wall. Sporonts from summer bees had only a thin single limiting membrane in some areas and evidence of endocytosis was sometimes seen in these. Sporonts from winter bees had branched tubular outpocketings from the wall. In sporoblasts, the development of the polar filament was closely associated with a network of dense structures interwoven with a system of tubules evidently of ER derivation; the Golgi complex was associated with this network.

Production of monoclonal antibodies specific for Eimeria tenella microgametocytes.

A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.

Detection of Nosema locustae (Microsporidia:Nosematidae) in frozen grasshoppers (Orthoptera:Acrididae) by using monoclonal antibodies.

We developed several monoclonal antibodies that recognized germinated and nongerminated spores and other developmental stages of Nosema locustae, a pathogen of grasshoppers, by using spore homogenate as antigen. The hybridomas were produced with standard techniques. Indirect fluorescence assay demonstrated recognition by several antibodies to the spore and extruded polar filament. Monoclonal antibody D10-9-11 recognized the spore wall in smears and frozen sections of infected grasshopper fat body tissue, whereas monoclonal antibody D55-12 also labeled the extruded polar filament. Immunoelectron microscopy showed that monoclonal antibody D10-9-11 recognized the spore and several developmental stages. Enzyme-linked immunosorbent assay showed that monoclonal antibody D10-9-11 did not cross-react with immunogen prepared from other grasshopper pathogens, including Vairimorpha sp., Malameba locustae, and entomopox virus from migratory grasshoppers, Melanoplus sanguinipes (F.).

Study of the tail fin rays of rainbow trout (Oncorhynchus mykiss) infected with the parasite Myxobolus cerebralis.

A light, transmission and scanning microscope study of the rays (lepidotrichia) forming the tail fin of rainbow trout (Oncorhynchus mykiss) fingerlings infected with the parasite Myxobolus cerebralis showed that triactinomyxon adherence to the tail fin of host fingerlings occurred 10 min after infection. After 2 h of exposure, it was possible to observe triactinomyxon spores in the epidermis. Although the characteristic symptoms of the disease, such as a black tail and a change in tail morphology, were observed, there was no attack against the tissue forming the tail fin rays and triactinomyxon spores were not observed inside the leptotrichial matrix at any stage, indicating that the spores do not reach the rays or that their observation was not possible under the conditions of the present study.

Histologic study of head cartilage degeneration in rainbow trout (Oncorhynchus mykiss) infected with the parasite Myxobolus cerebralis.

A light microscopy study of head cartilage tissue in rainbow trout alevins (Oncorhynchus mykiss) infected with the parasite Myxobolus cerebralis showed that, regardless of the presence or absence of whirling disease symptoms such as black tail and whirling swimming due to altered tail and spine morphology, some fish presented large amounts of spores lodged in the head after three months of infection. The spores were located in regions where the cartilage was extensively destroyed.

[Success of antegrade enemas in children with functional constipation]. / Efficacia di clisteri "anterogradi" in bambini con stipsi funzionale.

AIM: To assess the benefit of antegrade enemas via cecostomy in children with functional constipation unresponsive to medical treatment. METHODS: A retrospective chart review identified 19 children (12 male, age 10.4 +/- 4.3 yrs) who from 1998-2001 underwent placement of a cecostomy for administration of antegrade enemas. All children were neurologically normal, suffered from functional constipation; all had a history of using multiple medications, having daily soiling, and 65% had prior hospitalizations due to fecal impaction. Sixteen of 19 children had undergone colonic manometry which ruled out colonic inertia. One of 3 irrigation solutions: polyethylene glycol (65%), saline and glycerin solution mix (10%) and phosphate enema (25%) administered through the cecostomy everyday in 14 children or every other day in 5 children. We used a questionnaire to interview caregivers, a mean of 21.1 +/- 24.9 mo after cecostomy placement. RESULTS: In all patients antegrade enemas led to significant improvement of: bowel movements/wk (7.4 vs. 1.4, p < 0.001), soiling accidents/wk (1.1 vs. 6.1, p < 0.001), emotional health score (3.8 vs. 1.8, p < 0.001), overall health score (3.7 vs. 1.8, p < 0.001), number of medications for constipation (0.8 vs. 4.2, p < 0.001), number of missed school days/mo (1.3 vs. 10.5, p < 0.001), and number of physician office visits/yr (7.7 vs. 24.1, p < 0.002). Eight patients have been able to discontinue the use of the antegrade enemas within a mean of 19.9 +/- 14.2 mo after beginning treatment. CONCLUSION: Antegrade enemas are an alternative effective way for treating severe cases of functional constipation unresponsive to medical management.

Quality of life in patients with short bowel syndrome treated with the new glucagon-like peptide-2 analogue teduglutide--analyses from a randomised, placebo-controlled study.

BACKGROUND & AIMS: Short bowel syndrome (SBS)-intestinal failure (IF) patients have impaired quality of life (QoL) and suffer from the burden of malabsorption and parenteral support (PS). A phase III study demonstrated that treatment with teduglutide, a glucagon-like peptide 2 analogue, reduces PS volumes by 32% while maintaining oral fluid intake constant; placebo-treated patients had reduced PS by 21%, but oral fluid intake increased accordingly. As effects of teduglutide on QoL are unknown, they were investigated here. METHODS: QoL analyses from a double-blind, randomised Phase III study in 86 SBS-IF patients receiving teduglutide (0.05 mg/kg/day s.c.) or placebo over 24 weeks. At baseline and every 4 weeks, QoL was assessed using the validated SBS-QoL™ scale. RESULTS: PS reductions were associated with QoL improvements (ANCOVA, p = 0.0194, SBS-QoL per-protocol). Compared to baseline, teduglutide significantly improved the SBS-QoL™ total score and the score of 9 of 17 items at week 24. These changes were not significant compared to placebo. Teduglutide-treated patients with remaining small intestine >100 cm experienced more gastrointestinal adverse events (GI-AE), unfavourably affecting QoL. CONCLUSIONS: Overall, PS volume reductions were associated with improvements in SBS-QoL™ scores. The short observation period, imbalances in oral fluid intake in relation to PS reductions, large patient and effect heterogeneity and occurrence of GI-AE in a subgroup of teduglutide-treated patients may account for the inability to show statistically significant effects of teduglutide on SBS-QoL™ scores compared to placebo.

Distention of the colon is associated with initiation of propagated contractions in children.

The presence of high-amplitude propagating contractions (HAPCs) has been identified as a marker of colonic neuromuscular integrity. The physiologic mechanisms of HAPCs initiation have yet to be determined. Distention secondary to colonic filling has been hypothesized as physiologic initiator. The aim of this study was to study the effect of intraluminal balloon distention in the colon of children with defecatory disorders. Colonic manometry was performed with a polyethylene balloon situated at the proximal end of the catheter, which was placed in the most proximal colonic segment reached during colonoscopy. A stepwise pressure controlled distention of the balloon was performed using barostat computer (10-50 mmHg). Propagated contractions were defined as those that migrated over at least three recording sites. They were divided into HAPCs, amplitude >60 mmHg and low-amplitude propagating contractions (LAPCs), amplitude <60 mmHg. Children with spontaneous HAPCs or HAPCs after bisacodyl provocation were considered to have normal motility. Twenty children completed the study. Among the 14 children with normal colonic motility, balloon distention elicited HAPCs in four and LAPCs in 10 children. No HAPC were elicited in six children with abnormal motility and LAPCs were seen in four of them. The balloon-induced propagated contractions had similar characteristics as those occurring spontaneously and after bisacodyl provocation but the pressure needed to elicit them and their amplitude was inconsistent. These findings suggest that intraluminal distention can trigger propagated contractions in children. This mechanism of action for induction of propagated contractions is not as consistent as the motor response found in response to bisacodyl administration.

Childhood constipation: evaluation and treatment.

Constipation is common in children. It is estimated that between 5% and 10% of pediatric patients have constipation and/or encopresis. Constipation is the second most referred condition in pediatric gastroenterology practices, accounting for up to 25% of all visits. In this article, a practical approach is laid out for those not familiar with constipation in children. Emphasis is placed on the evaluation and management options that are available to the treating practitioner. The diagnosis of constipation requires careful history taking and interpretation. Diagnostic tests are not often needed and are reserved for those who are severely affected. The daily bowel habits of children are extremely susceptible to any changes in routine environment. Constipation and subsequent fecal retention behavior often begins soon after a child has experienced a painful evacuation. Childhood constipation can be very difficult to treat. It often requires prolonged support by physicians and parents, explanation, medical treatment, and, most important, the child's cooperation.

Development in cell cultures of Eimeria vermiformis Ernst, Chobotar and Hammond, 1971.

Development of Eimeria vermiformis from sporozoite to mature first-generation schizonts in cultured bovine kidney cells, Madin-Darby bovine kidney cells, and primary cultures of whole mouse embryos is described. Intracellular sporozoites were seen at 5 min, and for as long as 120 h after inoculation. Sporozoites were observed penetrating cells, with uninucleate trophozoites and immature schizonts with 2--6 nuclei first appearing 24 h after inoculation. Schizonts with 6 or more nuclei, as well as mature schizonts containing first-generation merozoites, were first seen between 36 and 48 h after inoculation of all 3 cell types used. The first indication of merozoite formation was determined by the appearance of small protuberances of cytoplasm at the periphery of schizonts. Merozoites began development at the periphery of schizonts and were later observed radiating from a central body of cytoplasm, 14--20 merozoites being formed. Some mature schizonts retained a small spherical residual body after merozoite formation was completed. After the rupture of schizonts, intracellular merozoites, which contained anterior and posterior refractile granules, were seen at 48, 72 and 96 h postinoculation. Merozoites were not seen entering or leaving cells. No further development was observed.

A procaryotic intracellular symbiont of the Great Salt Lake brine shrimp Artemia salina (L.).

Individuals of Artemia salina, the brine shrimp, were captured from the Great Salt Lake, a highly saline (330--340 g (see formula: solids content) terminal lake in Utah. Electron microscopy revealed the presence of intracellular procaryotic symbionts in the epithelial lining of the midgut. Fine structure and possible role of the symbiont are discussed.

Isolation and characterization of a linear plasmid from the entomopathogenic fungus Ascosphaera apis.

An extrachromosomal DNA plasmid was isolated from both mating types of the entomopathogenic fungus Ascosphaera apis and named pAaL. The subculture in which pAaL was first identified originated from mummified honey bee larvae from an apiary in Wyoming. Very similar, homologous plasmids were found in 9 out of 10 of isolates collected from diverse geographic locations. The plasmid is found inside the mitochondria, has the same buoyant density as mitochondrial DNA in bisbenzamide--CsCl gradients, and does not contain sequences homologous to either mitochondrial DNA or genomic DNA. The plasmid is linear, double-stranded, of 12 kilobase pair, and has a higher copy number than the mitochondrial DNA. Endonuclease and exonuclease digestions suggest that an inverted repeat is probably present at each terminus and that pAaL has two blocked 5' ends, probably due to the presence of terminal binding proteins. Restriction site data showed pAaL to be AT-rich. There were no apparent differences in the growth rate, culture appearance, and reproductive cycle of plasmid-bearing or plasmid-free A. apis isolates. pAaL was stably inherited in the plasmid-free strains, but it was lost in the progeny of crosses and reciprocal crosses between the plasmid-free strain and plasmid-bearing strains. The biological function of this plasmid has not yet been determined.

Esterase isozymes in a solitary bee, Megachile rotundata (Fab.): characterization, developmental multiplicity, and adult variability.

This study describes the biochemical characterization and genetic variation of cytosolic esterases in the alfalfa leafcutting bee, Megachile rotundata (Fab.). Esterase isozymes were separated by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing and characterized by inhibition with eserine sulfate, EDTA, paraoxon, and p-hydroxymercuribenzoate. Based on inhibition patterns and substrate specificity, there are major differences between adults and immature forms and more subtle differences between male and female adults. M. rotundata esterases are largely organophosphate sensitive and the two major adult allozymes were highly variable within the population examined. Differences in esterase expression between life stages with respect to niche and the occurrence of diploid males are discussed.