The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its “a” determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the “a” determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of “a” determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.
Coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) is a serious pest of coffee worldwide and a new invasive pest in Hawaii. Adult flat bark beetles, mainly Leptophloeus sp. (75%) and Cathartus quadricollis (21%) (Coleoptera: Laemophloeidae and Silvanidae, respectively), were found inside CBB-infested coffee beans on the tree and molecular marker studies confirmed feeding on CBB. Research was conducted to better understand the ecology of these predators and explore ways to increase their role in suppressing CBB populations in coffee. Laboratory feeding assays demonstrated the capacity for adult and larval flat bark beetles to feed on all CBB life stages. The predators are widely distributed in the coffee growing areas on the island of Hawaii, but feed mainly in dried coffee on the tree rather than in ripening berries where initial crop damage occurs. Berlese funnel extraction of flat bark beetles from dried beans on the tree indicated that predator numbers can be relatively high (up to 23 adult predators per 150 bean sample). C. quadricollis was not susceptible to infection by the fungal biopesticide Beauveria bassiana which is used for field control of CBB in coffee. Leptophloeus sp. and C. quadricollis can be raised easily on a diet of cracked corn and cornmeal. Augmentative releases of generalist flat bark beetle predators like Leptophloeus sp. and C. quadricollis may be a useful component of integrated pest management programs against coffee berry borer and other scolytine pests.
The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F 1 males but fertile F 1 females, a pattern consistent with Haldane's rule. Backcrossing F 1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than transdivergent genes, with no significant difference in the ratio of cis-and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species.
Coffee is one of the most economically valuable specialty crops for which Hawaii is famous. It is produced commercially on >6,900 acres across six islands by more than 1,470 growers. It has a raw crop value of $55.9 million, while the value-added benefits of coffee-related industries exceed $148.5 million (USDA, 2021). In addition to high product quality, Hawaii also has the distinction of being the last major coffee growing region that is free of coffee leaf rust (CLR), a highly damaging disease caused by the obligate parasitic fungus Hemileia vastatrix Berk. & Broome. On October 21, 2020, a coffee grower in Maui County, Hawaii reported the presence of coffee leaf rust-like symptoms on coffee plants (var. ‘Typica’) at their farm with many trees heavily defoliated. Foliar symptoms consisted of yellowish-orange, circular lesions that often coalesced. On abaxial surfaces, these lesions appeared powdery. Urediniospores were mostly reniform, (25)27-34(36) × (17)18-28(29) µm, strongly echinulated on the upper (convex) surface and smooth on the lower (concave) surface, with hyaline to pale yellow-orange walls. Urediniospores incubated in molecular grade water for 10 min at 95 oC served as template for PCR targeting the internal transcribed spacer region of fungi using primers ITS1/ITS2, ITS3/ITS4, and ITS1/ITS4 (White et al., 1990). Amplification products underwent direct Sanger-based sequencing. Following primer sequence trimming, the sequence reads were assembled using CAP3 (Huang and Madan, 1999) and deposited in GenBank (Accession MW228837). Blastn analysis revealed > 99% nucleotide identity with isolates of H. vastatrix from Mexico (eg. KX260251) and Brazil (eg. MF627828). A voucher material from which both the morphological and molecular assays were performed was deposited in the National Fungus Collection (BPI 924818). Subsequent surveys on the islands of Hawaii, Lanai, and Oahu revealed the presence of coffee plants with symptoms of coffee leaf rust. To confirm pathogenicity, urediniospores from a symptomatic plant growing in Holualoa, Hawaii, were collected in gelatin capsules using a G-R Electric Manufacturing Portable Vacuum Pump with a mini cyclone spore adapter. The concentration of spores was adjusted to 1 x 105 spores/ml in sterile water using a Brightline Hemocytometer. The spore suspension was brushed onto the abaxial side of leaves from two C. arabica var. ‘Typica’ plants using a camel hair paintbrush. Two control plants were mock-inoculated with sterile water. Plants were placed in a dark humid chamber set at 22 oC for 48 hours, after which it was adjusted to 12 hours light under cool white fluorescent lighting. After 20 days small, chlorotic spots were visible on the adaxial leaf surface and a few spots contained orange urediniospores on the abaxial surface. Lesions expanded by day 34, followed by necrosis of the center area of several leafspots and leaf abscission was observed at day 70. The experiment was conducted twice. H. vastatrix was confirmed by both morphological and molecular examination of urediniospores recovered from the observed lesions. All mock-inoculated control plants remained asymptomatic. To our knowledge, this is the first report of CLR on coffee in Hawaii. This discovery is of great concern since CLR threatens not only the yield and quality of Hawaii-grown coffee, but also the economic viability of this historic and culturally important industry. Statewide monitoring for CLR continues and further work is needed to mitigate the impact of this discovery through the development of short- and long-term management strategies.
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