Ultrastructure of the compound eye of the diploid female beetle,Xyleborus ferrugineus

Summary The compound eye of female (diploid) Xyleborus ferrugineus beetles was examined with scanning and transmission electron microscopy. The eye is emarginate, and externally consists of roughly 70–100 facets. Each ommatidium is composed of a thickly biconvex lenslet with about 50 electron dense and rare layers. The lens facet overlies a crystalline cone of the acone type which is roughly hourglass-shaped. Pigment cells envelop the entire ommatidium, and pigment granules also are abundant throughout the cytoplasm of the 8 retinular cells. The rhabdomeres of 2 centrally situated photoreceptor cells effectively fuse into a rhabdom that extends from the base of the crystalline cone deeply into the ommatidium. Six distal peripheral retinular cells encircle the 2 central cells, and their rhabdomeres join laterally to form a rhabdomeric ring around the central rhabdom. The rhabdom and rhabdomeric ring are effectively separated by the cytoplasm of the two central retinular cells which contains the usual organelles and an abundance of shielding pigment granules. Eight axons per ommatidium gather in a tracheae-less fascicle before exiting the eye through the fenestrate basement membrane. No tracheation was observed among the retinular cells. Each Semper cell of each observed crystalline cone contained an abundance of virus-like particles near the cell nucleus. The insect is laboratory reared, and the visual system seems very amenable to photoreceptor investigations.This research was supported by the Director of the Research Division, C.A.L.S., University of Wisconsin, Madison; and in part by research grant No. RR-00779 from the Division of Research Resources, National Institutes of Health and by funds from the Schoenleber Foundation, Milwaukee, WI to D.M.N.     

Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.The host immune response elicited by CD8⁺ cytotoxic T lymphocytes (CTLs) is a major contributor to viral control following human immunodeficiency virus type 1 (HIV-1) infection (6, 39), but antiviral pressure exerted by CTLs is diminished by the selection of escape mutations in targeted regions throughout the viral proteome (7, 18, 29, 35, 41, 45, 57). A comprehensive identification of HLA-associated viral polymorphisms has recently been achieved through population-based analyses of HIV-1 sequences and HLA class I types from different cohorts worldwide (3, 8, 13-15, 34, 43, 50, 56, 63). However, despite improved characterization of the sites and pathways of immune escape, effective ways to incorporate these findings into immunogen design remain an area of debate. A better understanding of the impact of escape mutations on viral fitness may provide novel directions for HIV-1 vaccines that are designed to attenuate pathogenesis.The development of innovative vaccine strategies that can overcome the extreme diversity of HIV is a key priority (4). One proposed approach is to target the most conserved T-cell epitopes, which presumably cannot escape from CTL pressure easily due to structural or functional constraints on the viral protein (55). Complementary approaches include the design of polyvalent and/or mosaic immunogens that incorporate commonly observed viral diversity (4, 38) or the specific targeting of vulnerable regions of the viral proteome that do escape but only at a substantial cost to viral replication capacity (RC) (1, 40). A chief target of such vaccine approaches is the major HIV-1 structural protein Gag, which is known to be highly immunogenic and to elicit CTL responses that correlate with the natural control of infection (22, 36, 66). Indeed, several lines of evidence support a relationship between the selection of CTL escape mutations and reduced HIV-1 fitness. These include the reversion of escape mutations following transmission to an HLA-mismatched recipient who cannot target the epitope (19, 24, 41) as well as reduced plasma viral load (pVL) set point following the transmission of certain escape variants from donors who expressed protective HLA alleles (17, 27). Notably, these in vivo observations have been made most often for variations within Gag that are attributed to CTL responses restricted by the protective alleles HLA-B*57 and -B*5801 (17, 19, 27, 41). Most recently, reduced in vitro RCs of clinical isolates and/or engineered strains encoding single or multiple escape mutations in Gag selected in the context of certain protective HLA alleles, including B*57, B*5801, B*27, and B*13, have been demonstrated (9, 10, 42, 53, 59, 62). Despite these efforts, the goal of a T-cell vaccine that targets highly conserved and attenuation-inducing sites is hampered by a lack of knowledge concerning the contribution of most escape mutations to HIV-1 fitness as well as a poor understanding of the relative influence of HLA on the viral RC at different stages of infection.The mutability of HIV-1 permits the generation of progeny viruses encoding compensatory mutations that restore normal protein function and/or viral fitness. Detailed studies have demonstrated that the in vitro RC of escape variants in human and primate immunodeficiency viruses can be enhanced by the addition of secondary mutations outside the targeted epitope (10, 20, 52, 59, 65). Thus, vaccine strategies aimed at attenuating HIV-1 must also consider, among other factors, the frequency, time course, and extent to which compensation might overcome attenuation mediated by CTL-induced escape. Despite its anticipated utility for HIV-1 vaccine design, systematic studies to examine the consequences of naturally occurring CTL escape and compensatory mutations on viral RC have not been undertaken.We have described previously an in vitro recombinant viral assay to examine the impact of Gag-Protease mutations on HIV-1 RC (47, 49). Gag and protease have been included in each virus to minimize the impact of sequence polymorphisms at Gag cleavage sites, which coevolve with changes in protease (5, 37). Using this approach, we have demonstrated that viruses derived from HIV-1 controllers replicated significantly less well than those derived from noncontrollers and that these differences were detectable at both the acute/early (49) and chronic (47) stages. Escape mutations in Gag associated with the protective HLA-B*57 allele, as well as putative compensatory mutations outside known CTL epitopes, contributed to this difference in RC (47). However, substantial variability was observed for viruses from controllers and noncontrollers, indicating that additional factors were likely to be involved. Benefits of this assay include its relatively high-throughput capacity as well as the fact that clinically derived HIV-1 sequences are used in their entirety. Thus, it is possible to examine a large number of “real-world” Gag-Protease sequences, to define an RC value for each one, and to identify sequences within the population of recombinant strains that are responsible for RC differences.Here, we use this recombinant virus approach to examine the contribution of HLA-associated immune pressure on Gag-Protease RC during acute/early (n = 66) and chronic (n = 803) infections in the context of naturally occurring HIV-1 subtype B isolates from untreated individuals. In a recent report (64), we employed this system to examine the Gag-Protease RC in a similar cohort of chronic HIV-1 subtype C-infected individuals. The results of these studies provide important insights into the roles of immune pressure and fitness constraints on HIV-1 evolution that may contribute to the rational design of an effective vaccine.     

Nondisjunction and isochromosome formation in the B chromosome of maize

Bianchi et al. (1961) found that sectored losses of B-translocation chromosomes occur at a significant rate during early development of the endosperm and sporophyte. The losses were attributed to nondisjunction of the chromomosome, since B type chromosomes are known to undergo nondisjunction at the second pollen mitosis. Sector formation was further analyzed in the present paper, using the translocation, TB-9b. It was found that losses of the B⁹ chromosome during early endosperm mitoses occur only if the 9^B chromosome is present. In addition, sectors are produced in the sporophyte only if the 9^B and B⁹ chromosomes are inherited from the male parent. Both of these findings suggest that nondisjunction is indeed responsible for the B⁹ losses (see text). However, cytological observation of sectored plants demonstrates that isochromosome formation, rather than nondisjunction, produces most B⁹ losses in the sporophyte. The conflicting results can be reconciled by assuming that the same basic event, perhaps stickiness of the B⁹ chromosome, produces nondisjunction at the second pollen mitosis and isochromosome formation in the developing sporophyte. Observation of the isochromosome in pachytene reveals that a heterochromatic region corresponding to the short arm of the normal B⁹ is missing. The normal B⁹ chromosome is, therefore, an acrocentric chromosome.     

Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens

Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-gamma was induced after stimulation with A. otitidis. Although the release of IFN-gamma started within 18 h after stimulation with A. otitidis, intracellular production of IFN-gamma was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.     

Establishment and characterization of murine macrophage hybrids

Proteose peptone-induced peritoneal macrophages from CBA/J (H-2k) mice have been fused to a hypoxanthine phosphoribosyltransferase-negative variant of the P388D1 (H-2d) murine macrophage cell line. Six hybrid clones were isolated following HAT selection and further characterized. Five of the six clones express class I antigens of both parental haplotypes by microelisa and by flow cytometric analysis. Class II antigen expression of both haplotypes was apparent following a 72-hr incubation of the hybrids with concanavalin A-stimulated rat spleen cell supernatant. However, I-Ad was expressed in all hybrids to a greater extent than I-Ak. Three clones with the highest level of I-Ak expression, E5, C2, and C4, were capable of antigen presentation to the I-Ak-restricted T-cell line, D10.G4.1. LPS induction of the hybrids resulted in a 2- to 15-fold increase in the amount of IL-1 produced relative to the P388D1 parent. Finally, in distinction to P388D1, all hybrids demonstrated increased Fc-mediated erythrophagocytosis of chromium-labeled antibody-coated erythrocytes. These murine macrophage hybrids appear stable and should serve as useful models in understanding the regulation of macrophage function.     

A quarter of a world away: female humpback whale moves 10,000 km between breeding areas

Fidelity of individual animals to breeding sites is a primary determinant of population structure. The degree and scale of philopatry in a population reflect the fitness effects of social facilitation, ecological adaptation and optimal inbreeding. Patterns of breeding-site movement and fidelity are functions of social structure and are frequently sex biased. We report on a female humpback whale (Megaptera novaeangliae) first identified by natural markings off Brazil that subsequently was photographed off Madagascar. The minimum travel distance between these locations is greater than 9800 km, approximately 4000 km longer than any previously reported movement between breeding grounds, more than twice the species' typical seasonal migratory distance and the longest documented movement by a mammal. It is unexpected to find this exceptional long-distance movement between breeding groups by a female, as models of philopatry suggest that male mammals move more frequently or over longer distances in search of mating opportunities. While such movement may be advantageous, especially in changeable or unpredictable circumstances, it is not possible to unambiguously ascribe causality to this rare observation. This finding illustrates the behavioural flexibility in movement patterns that may be demonstrated within a typically philopatric species.     

Effects of AeDNV infection on Aedes aegypti lifespan and reproduction

The effects of Aedes Densovirus (AeDNV) infections on survival, fertility, fecundity and vertical transmission in Aedes aegypti (Diptera: Culicidae) were measured in laboratories in Kiev, Ukraine and Colorado, USA and incorporated into a predictive model of the effects of AeDNV on vector capacity. Adult lifespan and daily survival were reduced in AeDNV infected mosquitoes. This effect was dependent on the dose of the virus. Infected females had decreased fecundity. The oviposition rate was less in infected females and the hatch rate declined in eggs laid by infected females. The amounts of AeDNV in infected females and the infection rate of their offspring were measured with real-time PCR. The average filial transmission rate was 70% and larval infection rates from infected females varied between 42 and 62%. Vertically infected larvae, and individual eggs contained 1 × 10⁵ AeDNV genome equivalents (geq). Modeling the effects of AeDNV infection on Ae. aegypti populations suggested a large decrease in the numbers of eggs, larvae, pupae, and adults arising from infected mothers and suggested that AeDNV treatment of larvae could cause up to a 76% reduction of infectious mosquito days.     

Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium.

Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine max and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant omega-6 and omega-3 desaturases but more than 48% similarity to cyanobacterial omega-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid omega-6 desaturase. The identity was supported by expression of the B. napus cDNA in cyanobacterium. Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 73 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 16:1(9c) fatty acid was converted primarily to 16:2(9c, 12) in vivo. Thus, the plant omega-6 desaturase, which utilizes 16:1(7c) in plants, can utilize 16:1(9c) in the cyanobacterium. The plastid and cytosolic homologs of plant omega-6 desaturases are much more distantly related than those of omega-3 desaturases.