Gusti Ngurah Mahardika

Comparative Phylogeography of the Coral Triangle and Implications for Marine Management

Extreme concentration of marine biodiversity and exploitation of marine resources in the Coral Triangle pose challenges to biogeographers and resource managers. Comparative phylogeography provides a powerful tool to test biogeographic hypotheses evoked to explain species richness in the Coral Triangle. It can also be used to delineate management units for marine resources. After about a decade of phylogeographical studies, patterns for the Coral Triangle are emerging. Broad connectivity in some species support the notion that larvae have maintained gene flow among distant populations for long periods. Other phylogeographic patterns suggest vicariant events resulting from Pleistocene sea level fluctuations, which have, at least occasionally, resulted in speciation. Divergence dates ranging back to the Miocene suggest that changing land configurations may have precipitated an explosion of species diversification. A synthesis of the marine phylogeographic studies reveals repeated patterns that corroborate hypothesized biogeographic processes and suggest improved management schemes for marine resources.

Dietary breadth is positively correlated with venom complexity in cone snails

BackgroundAlthough diet is believed to be a major factor underlying the evolution of venom, few comparative studies examine both venom composition and diet across a radiation of venomous species. Cone snails within the family, Conidae, comprise more than 700 species of carnivorous marine snails that capture their prey by using a cocktail of venomous neurotoxins (conotoxins or conopeptides). Venom composition across species has been previously hypothesized to be shaped by (a) prey taxonomic class (i.e., worms, molluscs, or fish) and (b) dietary breadth. We tested these hypotheses under a comparative phylogenetic framework using ecological data from past studies in conjunction with venom duct transcriptomes sequenced from 12 phylogenetically disparate cone snail species, including 10 vermivores (worm-eating), one molluscivore, and one generalist.ResultsWe discovered 2223 unique conotoxin precursor peptides that encoded 1864 unique mature toxins across all species, >90xa0% of which are new to this study. In addition, we identified two novel gene superfamilies and 16 novel cysteine frameworks. Each species exhibited unique venom profiles, with venom composition and expression patterns among species dominated by a restricted set of gene superfamilies and mature toxins. In contrast with the dominant paradigm for interpreting Conidae venom evolution, prey taxonomic class did not predict venom composition patterns among species. We also found a significant positive relationship between dietary breadth and measures of conotoxin complexity.ConclusionsThe poor performance of prey taxonomic class in predicting venom components suggests that cone snails have either evolved species-specific expression patterns likely as a consequence of the rapid evolution of conotoxin genes, or that traditional means of categorizing prey type (i.e., worms, mollusc, or fish) and conotoxins (i.e., by gene superfamily) do not accurately encapsulate evolutionary dynamics between diet and venom composition. We also show that species with more generalized diets tend to have more complex venoms and utilize a greater number of venom genes for prey capture. Whether this increased gene diversity confers an increased capacity for evolutionary change remains to be tested. Overall, our results corroborate the key role of diet in influencing patterns of venom evolution in cone snails and other venomous radiations.

Newcastle disease virus-vectored H7 and H5 live vaccines protect chickens from challenge with H7N9 or H5N1 avian influenza viruses

ABSTRACT Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that Newcastle disease virus (NDV)-vectored H7 (NDV-H7) and NDV-H5 vaccines are able to induce antibodies with high hemagglutination inhibition (HI) titers and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus-expressed H7 protein failed to protect chickens from H7N9 virus infection.

Molecular evidence for co-occurring cryptic lineages within the Sepioteuthis cf. lessoniana species complex in the Indian and Indo-West Pacific Oceans

AbstractThe big-fin reef squid, Sepioteuthis cf. lessoniana (Lesson 1930), is an important commodity species within artisanal and near-shore fisheries in the Indian and Indo-Pacific regions. While there has been some genetic and physical evidence that supports the existence of a species complex within S. cf. lessoniana, these studies have been extremely limited in scope geographically. To clarify the extent of cryptic diversity within S. cf. lessoniana, this study examines phylogenetic relationships using mitochondrial genes (cytochrome oxidase c, 16s ribosomal RNA) and nuclear genes (rhodopsin, octopine dehydrogenase) from nearly 400 individuals sampled from throughout the Indian, Indo-Pacific, and Pacific Ocean portions of the range of this species. Phylogenetic analyses using maximum likelihood methods and Bayesian inference identified three distinct lineages with no clear geographic delineations or morphological discriminations. Phylogeographic structure analysis showed high levels of genetic connectivity in the most widespread lineage, lineage C and low levels of connectivity in lineage B. This study provides significant phylogenetic evidence for cryptic lineages within this complex and confirms that cryptic lineages of S. cf. lessoniana occur in sympatry at both small and large spatial scales. Furthermore, it suggests that two closely related co-occurring cryptic lineages have pronounced differences in population structure, implying that underlying differences in ecology and/or life history may facilitate co-occurrence. Further studies are needed to assess the range and extent of cryptic speciation throughout the distribution of this complex. This information is extremely useful as a starting point for future studies exploring the evolution of diversity within Sepioteuthis and can be used to guide fisheries management efforts.n

Mapping of cross-reacting and serotype-specific epitopes on the VP3 structural protein of the infectious bursal disease virus (IBDV)

SummaryThe binding sites of a panel of monoclonal antibodies cross-reacting with the structural protein VP3 of the two serotypes of the infectious bursal disease virus (IBDV) could be mapped to four segments of the VP3 gene. Two of these antigenic domains also carry epitopes which are specific for one serotype only. Formation of the common or type-specific epitopes is in agreement with homologous or mismatching amino acid sequences yielding hydrophilic segments on the VP3 polypeptide. These antigenic patterns obtained by immunoblotting could be verified by a competitive ELISA.

Phylogenetic analysis and victim contact tracing of rabies virus from humans and dogs in Bali, Indonesia

SUMMARY The emergence of human and animal rabies in Bali since November 2008 has attracted local, national and international interest. The potential origin and time of introduction of rabies virus to Bali is described. The nucleoprotein (N) gene of rabies virus from dog brain and human clinical specimens was sequenced using an automated DNA sequencer. Phylogenetic inference with Bayesian Markov Chain Monte Carlo (MCMC) analysis using the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) v. 1.7.5 software confirmed that the outbreak of rabies in Bali was caused by an Indonesian lineage virus following a single introduction. The ancestor of Bali viruses was the descendant of a virus from Kalimantan. Contact tracing showed that the event most likely occurred in early 2008. The introduction of rabies into a large unvaccinated dog population in Bali clearly demonstrates the risk of disease transmission for government agencies and should lead to an increased preparedness and efforts for sustained risk reduction to prevent such events from occurring in future.

Genetic structure and signatures of selection in grey reef sharks (Carcharhinus amblyrhynchos)

With overfishing reducing the abundance of marine predators in multiple marine ecosystems, knowledge of genetic structure and local adaptation may provide valuable information to assist sustainable management. Despite recent technological advances, most studies on sharks have used small sets of neutral markers to describe their genetic structure. We used 5517 nuclear single-nucleotide polymorphisms (SNPs) and a mitochondrial DNA (mtDNA) gene to characterize patterns of genetic structure and detect signatures of selection in grey reef sharks (Carcharhinus amblyrhynchos). Using samples from Australia, Indonesia and oceanic reefs in the Indian Ocean, we established that large oceanic distances represent barriers to gene flow, whereas genetic differentiation on continental shelves follows an isolation by distance model. In Australia and Indonesia differentiation at nuclear SNPs was weak, with coral reefs acting as stepping stones maintaining connectivity across large distances. Differentiation of mtDNA was stronger, and more pronounced in females, suggesting sex-biased dispersal. Four independent tests identified a set of loci putatively under selection, indicating that grey reef sharks in eastern Australia are likely under different selective pressures to those in western Australia and Indonesia. Genetic distances averaged across all loci were uncorrelated with genetic distances calculated from outlier loci, supporting the conclusion that different processes underpin genetic divergence in these two data sets. This pattern of heterogeneous genomic differentiation, suggestive of local adaptation, has implications for the conservation of grey reef sharks; furthermore, it highlights that marine species showing little genetic differentiation at neutral loci may exhibit patterns of cryptic genetic structure driven by local selection.

Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010.

Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses isolated from chicken farms in Indonesia from 2008 to 2010 was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003. Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p<0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies.

An investigation of classical swine fever virus seroprevalence and risk factors in pigs in East Nusa Tenggara, eastern Indonesia

Classical swine fever virus (CSFV) is a highly infectious disease of pigs. It has had significant impacts on East Nusa Tenggara, eastern Indonesia since its introduction in 1997. In spite of its importance to this region, little is known about its seroprevalence and distribution, and pig-level and farmer-level factors that may have an impact on the serological status of an individual pig. To address this knowledge deficit, a cross-sectional seroprevalence survey was conducted in 2010 involving 2160 pigs and 805 farmers from four islands in the region. Farmer questionnaires and pig record forms were used to collect data about the farmers and pigs surveyed. Blood was collected from each pig to determine its CSFV serological status. Apparent and true prevalence were calculated for each island, district, subdistrict, and village surveyed. CSFV serological status was used as an outcome variable in mixed effects logistic regression analyses. Overall true CSFV seroprevalence was estimated at 17.5% (lower CI 16.0%; upper CI 19.5%). Seroprevalence estimates varied widely across the islands, districts, subdistricts, and villages. Manggarai Barat, a district on the western end of Flores Island, contained pigs that were positive for antibody to CSFV. This result was unexpected, as no clinical cases had been reported in this area. Older pigs and pigs that had been vaccinated for CSFV were more likely to test positive for antibody to CSFV. The final multivariable model accounted for a large amount of variation in the data, however much of this variation was explained by the random effects with less than 2% of the variation explained by pig age and pig CSFV vaccination status. In this study we documented the seroprevalence of CSFV across four islands in East Nusa Tenggara, eastern Indonesia. We also identified risk factors for the presence of antibody to CSFV. Further investigation is needed to understand why clinical CSFV has not been reported on the western end of Flores Island, and to identify additional risk factors that explain CSFV serological status to inform disease control strategies.

Phylogeography of the current rabies viruses in Indonesia

Rabies is a major fatal zoonotic disease in Indonesia. This study was conducted to determine the recent dynamics of rabies virus (RABV) in various areas and animal species throughout Indonesia. A total of 27 brain samples collected from rabid animals of various species in Bali, Sumatra, Kalimantan, Sulawesi, Java, and Flores in 2008 to 2010 were investigated. The cDNA of the nucleoprotein gene from each sample was generated and amplified by one-step reverse transcription-PCR, after which the products were sequenced and analyzed. The symmetric substitution model of a Bayesian stochastic search variable selection extension of the discrete phylogeographic model of the social network was applied in BEAST ver. 1.7.5 software. The spatial dispersal was visualized in Cartographica using Spatial Phylogenetic Reconstruction of Evolutionary Dynamics. We demonstrated inter-island introduction and reintroduction, and dog was found to be the only source of infection of other animals. Ancestors of Indonesian RABVs originated in Java and its descendants were transmitted to Kalimantan, then further to Sumatra, Flores, and Bali. The Flores descendent was subsequently transmitted to Sulawesi and back to Kalimantan. The viruses found in various animal species were transmitted by the dog.