Evidence that microgynes of Myrmica rubra ants are social parasites that attack old host colonies.

Ant microgynes are miniaturized queen forms found together with normal queens (macrogynes) in species occurring across the ant phylogeny. Their role is not yet fully understood: in some cases, they seem to be nonparasitic alternative reproductive morphs, in others incipient social parasites, and thus potential models for studying the evolution of social parasitism. Whether they are regarded as parasitic or not has traditionally been based on genetic differentiation from syntopic macrogynes and/or the queen/worker ratio of their offspring rather than measuring fitness traits. We confirmed previously reported genetic differentiation between microgynes and macrogynes of Myrmica rubra in a population studied for the first time. Further, we measured virulence and infectivity of M. rubra microgynes in a controlled laboratory experiment. Nests headed only by macrogynes (controls), only by microgynes, and naturally and artificially mixed nests were kept under identical conditions. We found reduction in worker numbers of both naturally and artificially mixed macrogyne/microgyne nests compared with controls, and strong reduction but also surprising variation in fitness of nests headed only by microgynes. Microgyne nests produced workers, males and microgynes. Microgynes did not themselves reproduce in artificially mixed nests, but reproduced most in naturally mixed nests that had lost their macrogyne queen. This, together with higher mortality of field-collected macrogyne queens from naturally infested colonies and greater estimated relative age of macrogyne queens in naturally infected nests, suggests that they preferentially exploit older host colonies. We conclude that M. rubra microgynes are intraspecific social parasites specialized on exploiting old host colonies.

Dispersal and gene flow in the rare, parasitic Large Blue butterfly Maculinea arion.

Dispersal is crucial for gene flow and often determines the long-term stability of meta-populations, particularly in rare species with specialized life cycles. Such species are often foci of conservation efforts because they suffer disproportionally from degradation and fragmentation of their habitat. However, detailed knowledge of effective gene flow through dispersal is often missing, so that conservation strategies have to be based on mark-recapture observations that are suspected to be poor predictors of long-distance dispersal. These constraints have been especially severe in the study of butterfly populations, where microsatellite markers have been difficult to develop. We used eight microsatellite markers to analyse genetic population structure of the Large Blue butterfly Maculinea arion in Sweden. During recent decades, this species has become an icon of insect conservation after massive decline throughout Europe and extinction in Britain followed by reintroduction of a seed population from the Swedish island of Öland. We find that populations are highly structured genetically, but that gene flow occurs over distances 15 times longer than the maximum distance recorded from mark-recapture studies, which can only be explained by maximum dispersal distances at least twice as large as previously accepted. However, we also find evidence that gaps between sites with suitable habitat exceeding ∼20km induce genetic erosion that can be detected from bottleneck analyses. Although further work is needed, our results suggest that M. arion can maintain fully functional metapopulations when they consist of optimal habitat patches that are no further apart than ∼10km.

Dynamic Wolbachia prevalence in Acromyrmex leaf-cutting ants: potential for a nutritional symbiosis.

Wolbachia are renowned as reproductive parasites, but their phenotypic effects in eusocial insects are not well understood. We used a combination of qrt-PCR, fluorescence in situ hybridization and laser scanning confocal microscopy to evaluate the dynamics of Wolbachia infections in the leaf-cutting ant Acromyrmex octospinosus across developmental stages of sterile workers. We confirm that workers are infected with one or two widespread wsp genotypes of Wolbachia, show that colony prevalence is always 100% and characterize two rare recombinant genotypes. One dominant genotype is always present and most abundant, whereas another only proliferates in adult workers of some colonies and is barely detectable in larvae and pupae. An explanation may be that Wolbachia genotypes compete for host resources in immature stages while adult tissues provide substantially more niche space. Tissue-specific prevalence of the two genotypes differs, with the rarer genotype being over-represented in the adult foregut and thorax muscles. Both genotypes occur extracellularly in the foregut, suggesting an unknown mutualistic function in worker ant nutrition. Both genotypes are also abundant in the faecal fluid of the ants, suggesting that they may have extended functional phenotypes in the fungus garden that the ants manure with their own faeces.

A phylogenetic revision of the Glaucopsyche section (Lepidoptera: Lycaenidae), with special focus on the Phengaris-Maculinea clade.

Despite much research on the socially parasitic large blue butterflies (genus Maculinea) in the past 40 years, their relationship to their closest relatives, Phengaris, is controversial and the relationships among the remaining genera in the Glaucopsyche section are largely unresolved. The evolutionary history of this butterfly section is particularly important to understand the evolution of life history diversity connected to food-plant and host-ant associations in the larval stage. In the present study, we use a combination of four nuclear and two mitochondrial genes to reconstruct the phylogeny of the Glaucopsyche section, and in particular, to study the relationships among and within the Phengaris-Maculinea species. We find a clear pattern between the clades recovered in the Glaucopsyche section phylogeny and their food-plant associations, with only the Phengaris-Maculinea clade utilising more than one plant family. Maculinea is, for the first time, recovered with strong support as a monophyletic group nested within Phengaris, with the closest relative being the rare genus Caerulea. The genus Glaucopsyche is polyphyletic, including the genera Sinia and Iolana. Interestingly, we find evidence for additional potential cryptic species within the highly endangered Maculinea, which has long been suspected from morphological, ecological and molecular studies.

Antibodies of the IgA type in intestinal plasma cells of germfree mice after oral or parenteral immunization with ferritin.

In adult germfree C(3)H mice immunized with horse spleen ferritin, either subcutaneously or intraperitoneally, plasma cells containing specific antibodies were found in lymph nodes and spleen and, in smaller numbers, also in the lamina propria of the intestine. In extraintestinal sites, these antiferritin-containing plasma cells were mainly of the IgM class after a single stimulation, and of the IgG(1) class after repeated stimulation. In the intestine, all the anti-ferritin-containing cells appeared to be of the IgA class. Circulating antibodies, after repeated stimulation, were for the major part IgG(1) and IgG(2). In germfree mice given ferritin in their drinking water, antiferritin-containing cells were abundant in the intestinal mucosa, much less numerous in the mesenteric lymph nodes, and extremely scarce in other lymphoid tissues. All these cells, whatever their location, appeared to belong exclusively to the IgA class. Similarly, all the circulating antibody in these animals was found to be IgA. These findings illustrate the role of the gut as a site of antibody synthesis, as well as its selective commitment to the production of antibodies of the IgA class.

Pretreatment, prediagnosis immunoglobulin, and alpha 1-antitrypsin levels in patients with bronchial carcinoma.

Sera from 148 patients suspected of having bronchial carcinoma were evaluated for concentrations of IgA, IgM, IgG, and alpha 1-antitrypsin (alpha 1-AT). Histologically, 137 tumor specimens obtained by bronchoscopy or surgical biopsy were diagnosed as squamous cell carcinoma (55%), adenocarcinoma (18%), undifferentiated small cell carcinoma (20%), or undifferentiated large cell carcinoma (7%). The tumor types of the remaining 11 patients were not identifiable from the records but were included as a separate group. Serum protein values were compared with those of a control group of 60 healthy adult volunteers. Regression analysis of age, race, and sex effects on the results of comparisons between test and control groups indicated that, with the possible exception of age and IgM, these three independent variables did not significantly influence the observations. Both serum IgA and alpha 1-AT were significantly elevated in all tumor groups when compared to the control levels. This was not the situation for serum IgG. Results for serum IgM were equivocal and depended on the statistical methods used. Although the serum immunoglobulin concentrations for the small cell carcinoma group were consistently lower than those for the other tumor cell types, no statistically significant difference existed between the five tumor groups in this regard.

Factors XWenatchee I and II: compound heterozygosity involving two variant proteins.

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10-20% of normal) and antigen (30-35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13,984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors XWenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

Amplification and cloning of the Mycobacterium tuberculosis dnaA gene.

To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.

Antibiotic susceptibilities and drug resistance in Moraxella (Branhamella) catarrhalis.

PURPOSE: To summarize current knowledge of drug susceptibility and mechanisms of drug resistance in Moraxella (Branhamella) catarrhalis. MATERIALS AND METHODS: The current medical literature was reviewed, with careful attention to recent studies of the BRO beta-lactamases. RESULTS: Although intrinsically resistant to a small group of drugs that included vancomycin and trimethoprim, acquired drug resistance in Branhamella catarrhalis was unknown in the early years of antimicrobial therapy. During 1976 to 1977, however, two previously unrecognized beta-lactamases appeared in this species almost simultaneously around the world. At the same time these enzymes, now known as BRO-1 and BRO-2, also appeared in two closely related commensal species of Moraxella. Within four years the BRO enzymes were found in up to 75 percent of B. catarrhalis in the United States where they provide low-level resistance to penicillin and ampicillin but not to most cephalosporins. The BRO genes appear to be chromosomal but are readily transferred by conjugation within the Moraxella genus. Resistance to aminoglycosides and trimethoprim-sulfamethoxazole has been reported from Spain, and resistance to both erythromycin and tetracycline has recently been described among United States isolates of B. catarrhalis. CONCLUSION: Despite this drug resistance, numerous oral and parenteral agents are available and appear useful for treatment of clinical disease, including amoxicillin/clavulanic acid, erythromycin, the tetracyclines, ciprofloxacin, and trimethoprim/sulfamethoxazole. Recent changes in drug resistance in this species suggest that continued monitoring of drug resistance in B. catarrhalis is needed.

Incidence of Branhamella catarrhalis in the sputa of patients with chronic lung disease.

The incidence of Branhamella catarrhalis in the respiratory tract of adults, especially in the United States, is not known. During the 30-month period from January 1983 to June 1985, 4180 sputum and endotracheal samples from patients in a hospital for chest diseases were evaluated. All samples were acceptable for Gram-stain analysis and/or culture based on published cellular criteria. Using primarily Gram-stain directed cultures, 220 isolates of B. catarrhalis were identified in 180 patients, being present in 5.3% of all sputum cultures and 11.5% of those positive for a pathogen. B. catarrhalis was the fourth most common pathogen identified. It was found in pure culture (124) and mixed culture (96), the latter usually in association with Haemophilus influenzae or Streptococcus pneumoniae. Of the 220 B. catarrhalis isolates, 158 (71.8%) were positive for beta-lactamase. The number and incidence of B. catarrhalis varied, with the organism being most prevalent during the winter months. Despite its frequent presence in sputum, B. catarrhalis was not recovered from pleural fluid or blood during the same period. This study demonstrates the frequent presence of B. catarrhalis in the sputum of adults with chronic lung disease, although the role of this organism as a pathogen was not determined.

Isoelectric focusing of beta-lactamases from sputum and middle ear isolates of Branhamella catarrhalis recovered in the United States.

Branhamella catarrhalis obtained from the sputum of 146 patients with lower respiratory tract disease and from middle ear fluids of 26 children with otitis media were evaluated for beta-lactamase activity and the enzymes were characterised by isoelectric focusing (IEF). 71% (103 of 146) of the sputum isolates and 77% (20 of 26) of the ear isolates produced beta-lactamase. By IEF, the beta-lactamases of 113 of 123 (92%) strains revealed patterns identical with the Ravasio type strain, having unique enzyme bands at pIs of 6.4 and 6.65. The remaining 10 isolates (8%) produced patterns similar to the 1908 type strain with a unique band of activity having a pI of 6.55. In addition, the 1908 types revealed a band of minor enzyme activity with a pI of 7.55 that was absent from the Ravasio types. All strains tested shared major enzyme bands with pIs of 5.1, 5.3, 5.55 and 6.1. These results indicate that the most common beta-lactamase(s) produced by clinical isolates of B. catarrhalis in the United States are similar to those produced by the Belgian Ravasio type strain.

Anergy in active pulmonary tuberculosis. A comparison between positive and negative reactors and an evaluation of 5 TU and 250 TU skin test doses.

Based on induration of less than 10 mm at 48 and 72 hours, 49 of 200 (25 percent) patients with active pulmonary tuberculosis failed to respond to intermediate (5TU) strength PPD. Distribution of reactors vs nonreactors was similar with regard to sex but not race, with more white subjects proving to be nonreactive. Additional comparisons between the two groups revealed significantly higher values in the responder population for age, in vitro lymphocyte response to PPD, total serum protein and serum IgG. Responsiveness of lymphocytes to phytohemagglutinin was also increased in this group, but only at lower mitogen concentrations. Alternatively, the nonresponder group demonstrated higher values for alpha-1 globulins. A group of nontuberculous patients similarly subjected to skin testing showed negative reactivity to 5 TU PPD at the much higher incidence of 152/173 (88 percent). The 49 patients with pulmonary tuberculosis and 152 nontuberculous patients who did not respond to 5 TU PPD were further tested with 250 TU PPD. In the group with tuberculosis, 30/49 (61.2 percent) demonstrated positive reactions, whereas only 55/152 (36.2 percent) nontuberculous patients showed induration equal to 10 mm or higher. Skin testing with the recall antigens TOE and SK-SD indicated that only 3/200 (1.5 percent) tuberculous patients could be classified as being anergic; 9/200 (4.5 percent) were specifically anergic to PPD and 7/200 (3.5 percent) demonstrated questionable anergy to PPD as judged by a 250 TU PPD response that was greater than 5 but less than 10 mm.

[(3)H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain.

[(3)H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT(1A) receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B(max) and affinity of [(3)H]LSD binding to the 5-HT(1A) binding site were significantly decreased. [(3)H]LSD in the presence of 40 nM WB4101 labeled the 5-HT(2) receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [(3)H]LSD binding to the 5-HT(1A) binding site, GTP produced no significant effect on either the B(max) or the K(D) of [(3)H]LSD binding to the 5-HT(2) binding site. Competition of 5-HT for [(3)H]LSD binding to the 5-HT(2) binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC(50). These binding characteristics are consistent with [(3)H]LSD acting as an antagonist at the 5-HT(2) receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [(3)H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT(1A) receptor subtype but as an antagonist at the 5-HT(2) receptor subtype in rat brain.

Selecting drug combinations for treatment of drug-resistant mycobacterial diseases.

Mixtures of antituberculosis drugs were evaluated for their in vitro effects on drug-resistant isolates of Mycobacterium tuberculosis and M. avium-intracellulare. The response of individual isolates to representative drug combinations was not always predictable from the results of single-drug sensitivity assays. For the case of M. tuberculosis, combinations of drugs were often bactericidal even under conditions where two or more drugs were without effect when tested singly. The more widely drug-resistant M. avium-intracellulare demonstrated increased growth inhibition when subcultured in the presence of single drugs, particularly rifampin and streptomycin. However, these conditions favored the selection of highly resistant strains. Alternatively, multiple drugs were often bacteriostatic; and under conditions where isolates demonstrated growth inhibition, the selection of highly drug-resistant strains was delayed. These results suggest a role for multiple-drug sensitivity assays in selecting drug combinations to be used in the treatment of drug-resistant mycobacterioses.

Induction of aryl hydrocarbon hydroxylase by lymphocytes from women taking oral contraceptives.

The peripheral blood lymphocytes (PBL) from two female subjects were assayed for AHH induction 40 days prior to and 30 days during ingestion of progesterone and estrogen analogues as oral contraceptives. Three habitual users of oral contraceptives were also studied. No in vitro inhibition of AHH induction was observed as a consequence of the use of these hormone analogues. Values obtained for enzyme activity suggest a slight increase in AHH induction resulting from the use of oral contraceptives. Further studies with larger numbers of subjects are required before the apparent increase in enzyme inducibility can be considered significant.

PIP: 2 healthy female volunteers were tested 3 times weekly to determine any effects of ingestion of a combined oral contraceptive (OC) agent on the inducibility of aryl hydrocarbon hydroxylase (AHH), an enzyme known to be readily synthesized by human lymphocytes in the presence of carcinogens. These 2 subjects were assayed 40 days before and 30 days during ingestion. 3 habitual users of OCs were also studied. In the absence of OCs, the 2 highest values occurring at or immediately following the approximate time of ovulation. In the presence of a lower dose OC (.3 mg ethinyl estradiol and .3 norgestrel), the daily levels of AHH inducibility followed a cyclical pattern very similar to that found in the absence of OCs, with a peak at Day 17. On higher dosage (.5 mg ethinyl estradiol and .5 mg norgestrel), a greater degree of variability was noticed, but the overall inducibility pattern followed the same general trend. Hence, no in vitro inhibition of AHH induction was observed as a consequence of using hormone analogs; but the values, shown graphically, obtained for the enzyme activity suggested a slight increase in AHH induction resulting from the OC use. Further, larger studies are called for.

A tuberculosis hotline in Tyler: a Texas resource for primary health care providers for the control of infectious diseases.

The Center for Pulmonary and Infectious Disease Control (CPIDC), located on the campus of The University of Texas Health Center in Tyler, manages a toll-free infectious disease consultation hotline advertised to public and private physicians and to health care agencies throughout the state. From January 1994 through December 1996, as part of a statewide initiative to curb an unprecedented increase in the incidence of tuberculosis observed since 1985, a concentrated effort was made to solicit health care providers for consultation requests that involved the diagnosis and management of tuberculosis, in particular, drug-resistant varieties. During that period, 3447 calls were made to the CPIDC by 1682 physicians and nurses. While most of the calls originated from 4 major urban areas plus health care facilities along the border, calls were received from more than half of all the counties in Texas. The value of providing an infectious disease consultation service, readily available, without charge, to all members of the health care community is discussed.