Drosophila Embryos as Model Systems for Monitoring Bacterial Infection in Real Time

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.     

EFFECTS OF INTRINSIC AND EXTRINSIC FACTORS ON POPULATION FRAGMENTATION IN THREE SPECIES OF NORTH AMERICAN MINNOWS (TELEOSTEI: CYPRINIDAE)

Geographic patterns of genetic variation (mitochondrial DNA [mtDNA] and allozymes) were used to examine effects of intrinsic characteristics (e.g., vagility, habitat specificity, and reproductive behaviors) and extrinsic factors (e.g., climatic and geological history) on population fragmentation. The three species of cyprinid fishes examined (Tiaroga cobitis, Meda fulgida, and Agosia chrysogaster) occupied similar historical ranges within the lower Colorado River drainage, but differ in intrinsic characteristics conducive to population fragmentation. Relationships among populations were similar across species, reflecting common historical influences, but results indicate the distribution of variation among species is strongly affected by intrinsic characteristics. Variation within two species (T. cobitis and M. fulgida) is subdivided among populations, suggesting little gene flow among rivers. In contrast, similarity of A. chrysogaster populations throughout the Gila River drainage supports the hypothesis that levels of gene flow are high for this species. Levels of mtDNA divergence were much higher than expected for both T. cobitis and A. chrysogaster suggesting long-term isolation of geographic regions. These results indicate that both long-term and short-term extrinsic factors have shaped basic patterns of variation within these fishes; however, the intrinsic characteristics of each species have strongly affected the population genetic structure of these fishes.     

Modification of respiratory center output in the unanesthetized fetal sheep "in utero".

Diaphragmatic electromyographic activity, tracheal and amniotic fluid pressures, lung liquid flow, and carotid and jugular venous pressures were measured on eight fetal lambs who survived for periods of 9-43 days postoperatively. The fetal gestational age ranged from 98 to 113 days at operation. Respiratory center output of the fetus as indicated by electromyographic activity was modified by the following stimuli. It was suppressed by anesthesia and fetal hypoxia (Pao2 = 12 mmHg), tonically reduced by lung inflation, and stimulated by cyanide injections (150-600 mug) into the fetal jugular vein. Neuromuscular transmission to the diaphragm was blocked with d-tubocurarine (0.2-0.6 mg). These experiments indicate that central and motor pathways to the diaphragm are sufficiently mature by 101 days in the fetal sheep to permit their output to be modified by chemical and mechanical stimuli.     

Effective size,census size,and genetic monitoring of the endangered razorback sucker, <Emphasis Type="Italic">Xyrauchen texanus</Emphasis>

For the past several decades the Lake Mohave population of the federally endangered razorback sucker, Xyrauchen texanus, has had no natural recruitment because of intensive predation on larvae by non-native fishes. In response to impending extirpation, a repatriation program was implemented where larval razorbacks are collected from the wild following natural spawning, reared in protective custody, and then repatriated at a much larger size. In this study, we estimated annual (N _bf) and generational female effective size (N _ef) of the spawning stock by characterizing temporal genetic changes in mtDNA among larval cohorts, and then compared these estimates to the estimated number of reproductively capable (adult) females in Lake Mohave (N _f). Razorback suckers have life history and mortality schedules that could yield values of N _ef/N _f as low as 10⁻⁵ due to match–mismatch recruitment (or the “Hedgecock effect”) that increases variance in reproductive success. Average N _bf was estimated to be 160, N _ef was 706, and the ratio N _ef/N _f was 0.29 and 0.38 for arithmetic and harmonic mean N _f, respectively. Our findings indicate that (i) larval sampling in Lake Mohave sufficiently encompasses temporal and spatial variation within annual larval cohorts as to be representative of the spawning stock, (ii) roughly 3–16% (about 8% on average) of adult females contribute genetically to larval cohorts each year, and (iii) repatriated fishes appear to be contributing genetically to larval cohorts. Simultaneous genetic and demographic monitoring offers insights that neither approach can provide alone into effects of population decline and management practices in this species.