Evolutionary history of orangutan plasmodia revealed by phylogenetic analysis of complete mtDNA genomes and new biogeographical divergence dating calibration models.

During the past 15 years, researchers have shown a renewed interest in the study of the Plasmodium parasites that infect orangutans. Most recently, studies examined the phylogenetic relationships and divergence dates of these parasites in orangutans using complete mitochondrial DNA genomes. Questions regarding the dating of these parasites, however, remain. In the present study, we provide a new calibration model for dating the origins of Plasmodium parasites in orangutans using a modified date range for the origin of macaques in Asia. Our Bayesian phylogenetic analyses of complete Plasmodium sp. mitochondrial DNA genomes inferred two clades of plasmodia in orangutans (Pongo 1 and Pongo 2), and that these clades likely represent the previously identified species Plasmodium pitheci and Plasmodium silvaticum. However, we cannot identify which Pongo clade is representative of the morphologically described species. The most recent common ancestor of both Pongo sp. plasmodia, Plasmodium. hylobati, and Plasmodium. inui dates to 3-3.16 million years ago (mya) (95% highest posterior density [HPD]: 2.09-4.08 mya). The Pongo 1 parasite diversified 0.33-0.36 mya (95% HPD: 0.12-0.63), while the Pongo 2 parasite diversified 1.15-1.22 mya (95% HPD: 0.63-1.82 mya). It now seems likely that the monkey Plasmodium (P. inui) is the result of a host switch event from the Pongo 2 parasite to sympatric monkeys, or P. hylobati. Our new estimates for the divergence of orangutan malaria parasites, and subsequent diversification, are all several hundred thousand years later than previous Bayesian estimates.

The impact of baculovirus challenge on immunity: The effect of dose and time after infection.

Understanding how hosts respond to pathogen attack is crucial to disease management. The response of a host can be particularly important if hosts have to defend against multiple pathogens which could either benefit from or be suppressed by prior pathogen exposure. Insect defence against viruses is less well understood than responses to other entomopathogens and much of the information available relates to in vitro studies and model systems. Baculoviruses are natural pathogens of insects, particularly Lepidoptera, and have been well-studied in terms of their ecology, pest control potential and molecular biology. In order to examine how an insect reacts to baculovirus challenge, we measured components of the cellular and humoral immune response of the cabbage looper Trichoplusia ni to Trichoplusia ni SNPV, a narrow-host range nucleopolyhedrovirus (NPV), over four doses and three times after pathogen challenge (18, 42 and 90 h). We found that total haemocyte numbers peaked at 42 h post-exposure at all doses, and declined linearly with increasing dose after the 18 h time point. Two immune-related enzymes, phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD), showed very different responses. PO levels were lowest at the 42 h time point and were not influenced by virus dose when each time point was examined separately. GLD levels declined over time but they interacted with virus dose in a non-linear manner, such that there was an increase in levels at intermediate virus doses after 18 h, no effect at 42 h, and then declined as infection progressed at 90 h post-infection. These data suggest that baculoviruses can rapidly infect haemocytes (or cause a reduction in their numbers) in a dose-dependent manner once the infection is systemic, likely reducing the ability of the host to counter subsequent infections. However, the data do not support a direct role for PO in defence against baculoviruses. Whether GLD plays a role in virus defence is still unclear.

The synergistic effects of insecticidal essential oils and piperonyl butoxide on biotransformational enzyme activities in Aedes aegypti (Diptera: Culicidae).

The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and beta-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil + PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5-25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 +/- 1.8% over controls. The essential oils alone reduced GST activity by 3-20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58-76% and reduced GST activity by 3-85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti.

Plant terpenoids: acute toxicities and effects on flight motor activity and wing beat frequency in the blow fly Phaenicia sericata.

We evaluated the acute toxicities and the physiological effects of plant monoterpenoids (eugenol, pulegone, citronellal and alpha-terpineol) and neuroactive insecticides (malathion, dieldrin and RH3421) on flight muscle impulses (FMI) and wing beat signals (WBS) of the blow fly (Phaenicia sericata). Topically-applied eugenol, pulegone, citronellal, and alpha-terpineol produced neurotoxic symptoms, but were less toxic than malathion, dieldrin, or RH3421. Topical application of eugenol, pulegone, and citronellal reduced spike amplitude in one of the two banks of blow fly dorsolongitudinal flight muscles within 6-8 min, but with citronellal, the amplitude of FMIs reverted to a normal pattern within 1 hr. In contrast to pulegone and citronellal, where impulse frequency remained relatively constant, eugenol caused a gradual increase, then a decline in the frequency of spikes in each muscle bank. Wing beating was blocked permanently within 6-7 min of administering pulegone or citronellal and within 16 mins with eugenol. alpha-Terpineol-treated blow flies could not beat their wings despite normal FMI patterns. The actions of these monoterpenoids on blow fly flight motor patterns are discussed and compared with those of dieldrin, malathion, RH3421, and a variety of other neuroactive substances we have previously investigated in this system. Eugenol, pulegone and citronellal readily penetrate blow fly cuticle and interfere with flight muscle and/or central nervous function. Although there were differences in the effects of these compounds, they mainly depressed flight-associated responses, and acted similarly to compounds that block sodium channels and facilitate GABA action.

Differential gene expression from midguts of refractory and susceptible lines of the mosquito, Aedes aegypti, infected with Dengue-2 virus.

Suppressive subtractive hybridization was used to evaluate the differential expression of midgut genes of feral populations of Aedes aegypti (Diptera: Culicidae) from Colombia that are naturally refractory or susceptible to Dengue-2 virus infection. A total of 165 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries. The analysis showed a higher number of differentially expressed genes in the susceptible Ae. aegypti individuals than the refractory mosquitoes. The functional annotation of ESTs revealed a broad response in the susceptible library that included immune molecules, metabolic molecules and transcription factors. In the refractory strain, there was the presence of a trypsin inhibitor gene, which could play a role in the infection. These results serve as a template for more detailed studies aiming to characterize the genetic components of refractoriness, which in turn can be used to devise new approaches to combat transmission of dengue fever.

Generation and characterization of the antibacterial activity of a novel hybrid antimicrobial peptide comprising functional domains from different insect cecropins.

The search for new antimicrobial compounds involves finding novel sources of chemotherapeutic compounds or manipulating and combining structures from existing molecules. Small antimicrobial peptides (AMPs) are components of innate immune defenses characterized in greatest detail in insect-derived AMPs. We have generated hybrid AMPs (hAMPs) by combining functional motifs from different insect AMPs as a proof of principle that we can generate molecules with lower minimum inhibitory concentrations, and with different activity and target specificity than either parent molecule. A two-helix, cecropin-like hAMP was created by linking the N-terminal alpha helix of cecropin A from Aedes aegypti to the C-terminal alpha helix of cecropin A1 from Drosophila melanogaster. This molecule exhibits antibacterial activity at sub-micromolar concentrations with a target specificity that differs from either parent molecule. Antibacterial activity of the hybrid molecule was found to be greater against Gram-negative than Gram-positive bacteria. No hemolysis was observed in sheep red blood cells exposed to concentrations up to 50 micromol/L, suggesting the peptide is not detrimental to eukaryotic cells.

Larvicidal and oviposition-altering activity of monoterpenoids, trans-anithole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae).

BACKGROUND: Aedes aegypti L. is the major vector of dengue fever and dengue hemorrhagic fever. In an effort to find effective tools for control programs to reduce mosquito populations, the authors assessed the acute toxicities of 14 monoterpenoids, trans-anithole and the essential oil of rosemary against different larval stages of Ae. aegypti. The potential for piperonyl butoxide (PBO) to act as a synergist for these compounds to increase larvicidal activity was also examined, and the oviposition response of gravid Ae. aegypti females to substrates containing these compounds was evaluated in behavioral bioassays. RESULTS: Pulegone, thymol, eugenol, trans-anithole, rosemary oil and citronellal showed high larvicidal activity against all larval stages of Ae. aegypti (LC(50) values 10.3-40.8 mg L(-1)). The addition of PBO significantly increased the larvicidal activity of all test compounds (3-250-fold). Eugenol, citronellal, thymol, pulegone, rosemary oil and cymene showed oviposition deterrent and/or repellent activities, while the presence of borneol, camphor and beta-pinene increased the number of eggs laid in test containers. CONCLUSIONS: This study quantified the lethal and sublethal effects of several phytochemical compounds against all larval stages of Aedes aegypti, providing information that ultimately may have potential in mosquito control programs through acute toxicity and/or the ability to alter reproductive behaviors.

Rhodnius prolixus: identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization.

We report the identification of immune-related molecules from the fat body, and intestine of Rhodnius prolixus, an important vector of Chagas disease. Insects were challenged by introducing pathogens or Trypanosoma cruzi, the parasite that causes Chagas disease, into the hemocoel. RNA from intestines, or fat body were isolated 24h after stimulation. We used suppressive subtractive hybridization to identify immune-related genes, generated three subtracted libraries, sequenced the clones and assembled the sequences. The functional annotation revealed expressed sequence tags (ESTs) generated in response to various stimuli in all tissues, and included pathogen recognition molecules, regulatory molecules, and effector molecules.

Bayesian inference reveals ancient origin of simian foamy virus in orangutans.

Simian foamy viruses (SFVs) infect most nonhuman primate species and appears to co-evolve with its hosts. This co-evolutionary signal is particularly strong among great apes, including orangutans (genus Pongo). Previous studies have identified three distinct orangutan SFV clades. The first of these three clades is composed of SFV from P. abelii from Sumatra, the second consists of SFV from P. pygmaeus from Borneo, while the third clade is mixed, comprising an SFV strain found in both species of orangutan. The existence of the mixed clade has been attributed to an expansion of P. pygmaeus into Sumatra following the Mount Toba super-volcanic eruption about 73,000years ago. Divergence dating, however, has yet to be performed to establish a temporal association with the Toba eruption. Here, we use a Bayesian framework and a relaxed molecular clock model with fossil calibrations to test the Toba hypothesis and to gain a more complete understanding of the evolutionary history of orangutan SFV. As with previous studies, our results show a similar three-clade orangutan SFV phylogeny, along with strong statistical support for SFV-host co-evolution in orangutans. Using Bayesian inference, we date the origin of orangutan SFV to >4.7 million years ago (mya), while the mixed species clade dates to approximately 1.7mya, >1.6 million years older than the Toba super-eruption. These results, combined with fossil and paleogeographic evidence, suggest that the origin of SFV in Sumatran and Bornean orangutans, including the mixed species clade, likely occurred on the mainland of Indo-China during the Late Pliocene and Calabrian stage of the Pleistocene, respectively.

Detailed phylogenetic analysis of primate T-lymphotropic virus type 1 (PTLV-1) sequences from orangutans (Pongo pygmaeus) reveals new insights into the evolutionary history of PTLV-1 in Asia.

While human T-lymphotropic virus type 1 (HTLV-1) originates from ancient cross-species transmission of simian T-lymphotropic virus type 1 (STLV-1) from infected nonhuman primates, much debate exists on whether the first HTLV-1 occurred in Africa, or in Asia during early human evolution and migration. This topic is complicated by a lack of representative Asian STLV-1 to infer PTLV-1 evolutionary histories. In this study we obtained new STLV-1 LTR and tax sequences from a wild-born Bornean orangutan (Pongo pygmaeus) and performed detailed phylogenetic analyses using both maximum likelihood and Bayesian inference of available Asian PTLV-1 and African STLV-1 sequences. Phylogenies, divergence dates and nucleotide substitution rates were co-inferred and compared using six different molecular clock calibrations in a Bayesian framework, including both archaeological and/or nucleotide substitution rate calibrations. We then combined our molecular results with paleobiogeographical and ecological data to infer the most likely evolutionary history of PTLV-1. Based on the preferred models our analyses robustly inferred an Asian source for PTLV-1 with cross-species transmission of STLV-1 likely from a macaque (Macaca sp.) to an orangutan about 37.9-48.9kya, and to humans between 20.3-25.5kya. An orangutan diversification of STLV-1 commenced approximately 6.4-7.3kya. Our analyses also inferred that HTLV-1 was first introduced into Australia ~3.1-3.7kya, corresponding to both genetic and archaeological changes occurring in Australia at that time. Finally, HTLV-1 appears in Melanesia at ~2.3-2.7kya corresponding to the migration of the Lapita peoples into the region. Our results also provide an important future reference for calibrating information essential for PTLV evolutionary timescale inference. Longer sequence data, or full genomes from a greater representation of Asian primates, including gibbons, leaf monkeys, and Sumatran orangutans are needed to fully elucidate these evolutionary dates and relationships using the model criteria suggested herein.

The origin of malarial parasites in orangutans.

BACKGROUND: Recent findings of Plasmodium in African apes have changed our perspectives on the evolution of malarial parasites in hominids. However, phylogenetic analyses of primate malarias are still missing information from Southeast Asian apes. In this study, we report molecular data for a malaria parasite lineage found in orangutans. METHODOLOGY/PRINCIPAL FINDINGS: We screened twenty-four blood samples from Pongo pygmaeus (Kalimantan, Indonesia) for Plasmodium parasites by PCR. For all the malaria positive orangutan samples, parasite mitochondrial genomes (mtDNA) and two antigens: merozoite surface protein 1 42 kDa (MSP-1(42)) and circumsporozoite protein gene (CSP) were amplified, cloned, and sequenced. Fifteen orangutans tested positive and yielded 5 distinct mitochondrial haplotypes not previously found. The haplotypes detected exhibited low genetic divergence among them, indicating that they belong to one species. We report phylogenetic analyses using mitochondrial genomes, MSP-1(42) and CSP. We found that the orangutan malaria parasite lineage was part of a monophyletic group that includes all the known non-human primate malaria parasites found in Southeast Asia; specifically, it shares a recent common ancestor with P. inui (a macaque parasite) and P. hylobati (a gibbon parasite) suggesting that this lineage originated as a result of a host switch. The genetic diversity of MSP-1(42) in orangutans seems to be under negative selection. This result is similar to previous findings in non-human primate malarias closely related to P. vivax. As has been previously observed in the other Plasmodium species found in non-human primates, the CSP shows high polymorphism in the number of repeats. However, it has clearly distinctive motifs from those previously found in other malarial parasites. CONCLUSION: The evidence available from Asian apes indicates that these parasites originated independently from those found in Africa, likely as the result of host switches from other non-human primates.

Effects of the essential oil constituent thymol and other neuroactive chemicals on flight motor activity and wing beat frequency in the blowfly Phaenicia sericata.

BACKGROUND: The effects were evaluated of the plant terpenoid thymol and eight other neuroactive compounds on flight muscle impulses (FMIs) and wing beat frequency (WBF) of tethered blowflies (Phaenicia sericata Meig.). RESULTS: The electrical activity of the dorsolongitudinal flight muscles was closely linked to the WBF of control insects. Topically applied thymol inhibited WBF within 15-30 min and reduced FMI frequency. Octopamine and chlordimeform caused a similar, early-onset bursting pattern that decreased in amplitude with time. Desmethylchlordimeform blocked wing beating within 60 min and generated a profile of continuous but lower-frequency FMIs. Fipronil suppressed wing beating and induced a pattern of continuous, variable-frequency spiking that diminished gradually over 6 h. Cypermethrin- and rotenone-treated flies had initial strong FMIs that declined with time. In flies injected with GABA, the FMIs were generally unidirectional and frequency was reduced, as was seen with thymol. CONCLUSIONS: Thymol readily penetrates the cuticle and interferes with flight muscle and central nervous function in the blowfly. The similarity of the action of thymol and GABA suggests that this terpenoid acts centrally in blowflies by mimicking or facilitating GABA action.