Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species.

Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended

Using a Near-Infrared Spectrometer to Estimate the Age of Anopheles Mosquitoes Exposed to Pyrethroids

We report on the accuracy of using near-infrared spectroscopy (NIRS) to predict the age of Anopheles mosquitoes reared from wild larvae and a mixed age-wild adult population collected from pit traps after exposure to pyrethroids. The mosquitoes reared from wild larvae were estimated as <7 or ≥7 d old with an overall accuracy of 79%. The age categories of Anopheles mosquitoes that were not exposed to the insecticide papers were predicted with 78% accuracy whereas the age categories of resistant, susceptible and mosquitoes exposed to control papers were predicted with 82%, 78% and 79% accuracy, respectively. The ages of 85% of the wild-collected mixed-age Anopheles were predicted by NIRS as ≤8 d for both susceptible and resistant groups. The age structure of wild-collected mosquitoes was not significantly different for the pyrethroid-susceptible and pyrethroid-resistant mosquitoes (P = 0.210). Based on these findings, NIRS chronological age estimation technique for Anopheles mosquitoes may be independent of insecticide exposure and the environmental conditions to which the mosquitoes are exposed

Effect of Serotype and Strain Diversity on Dengue Virus Replication in Australian Mosquito Vectors

Dengue virus (DENV) is the most important mosquito-borne viral pathogen of humans, comprising four serotypes (DENV-1 to -4) with a myriad of genotypes and strains. The kinetics of DENV replication within the mosquito following ingestion of a blood meal influence the pathogen’s ability to reach the salivary glands and thus the transmission potential. The influence of DENV serotype and strain diversity on virus kinetics in the two main vector species, Aedes aegypti and Ae. albopictus, has been poorly characterized. We tested whether DENV replication kinetics vary systematically among serotypes and strains, using Australian strains of the two vectors. Mosquitoes were blood fed with two strains per serotype, and sampled at 3, 6, 10 and 14-days post-exposure. Virus infection in mosquito bodies, and dissemination of virus to legs and wings, was detected using qRT-PCR. For both vectors, we found significant differences among serotypes in proportions of mosquitoes infected, with higher numbers for DENV-1 and -2 versus other serotypes. Consistent with this, we observed that DENV-1 and -2 generally replicated to higher RNA levels than other serotypes, particularly at earlier time points. There were no significant differences in either speed of infection or dissemination between the mosquito species. Our results suggest that DENV diversity may have important epidemiological consequences by influencing virus kinetics in mosquito vectors

Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species.

BACKGROUND: Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. METHODS: Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. RESULTS: The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. CONCLUSION: RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended

Near-infrared spectroscopy as a complementary age grading and species identification tool for African malaria vectors

Near-infrared spectroscopy (NIRS) was recently applied to age-grade and differentiate laboratory reared Anopheles gambiae sensu strico and Anopheles arabiensis sibling species of Anopheles gambiae sensu lato complex. In this study, we report further on the accuracy of this tool for simultaneously estimating the age class and differentiating the morphologically indistinguishable An. gambiae s.s. and An. arabiensis from semi-field releases and wild populations. Nine different ages (1, 3, 5, 7, 9, 11, 12, 14, 16 d) of An. arabiensis and eight different ages (1, 3, 5, 7, 9, 10, 11, 12 d) of An. gambiae s.s. maintained in 250 × 60 × 40 cm cages within a semi-field large-cage system and 105 wild-caught female An. gambiae s.l., were included in this study. NIRS classified female An. arabiensis and An. gambiae s.s. maintained in semi-field cages as <7 d old or ≥7 d old with 89% (n = 377) and 78% (n = 327) accuracy, respectively, and differentiated them with 89% (n = 704) accuracy. Wild caught An. gambiae s.l. were identified with 90% accuracy (n = 105) whereas their predicted ages were consistent with the expected mean chronological ages of the physiological age categories determined by dissections. These findings have importance for monitoring control programmes where reduction in the proportion of older mosquitoes that have the ability to transmit malaria is an important outcome

Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species

BACKGROUND: Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. METHODS: Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. RESULTS: The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. CONCLUSION: RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended

Unraveling the insights into phage endolysin association

In view of the abundacy of phages (1), even rare phage-induced events are frequent at the global level. They have a staggering ecological impact on the bacterial population and in the evolution of bacterial genomic structure upon virus-host interactions, acting as agents in the recycling of organic matter and presenting a valuable tool in molecular biology and epidemiology. Th focus on genomic research have revealed information on open reading frames of proteins of interest (2). Increasing interest has been given to phage (endo )lysins in molecular biology, biotechnology and medicine. Lysins are phage lytic enzymes that break down the peptidoglycan of the bacterial cell wall at the terminal stage of the phage reproduction cycle, to release the phage progeny with the consequent death of the bacterial cells (3). Despite the increasing number of genomes in Genbank:, no effort has been made so far to understand the relation between lysins and their phage family and host species, presenting challenges in their annotation, comparative analysis, and representation. The almost 700 complete phage genomes deposited in the NCBI database were searched for the presence of lysins by making use of the pfam ( 4) identified domains and BLAST comparison of putative/unidentified complete genome against known lysins. In approximately 5% of the phage genomes it was not possible to identify any lysin. The identified enzymes were used to construct a phylogenetic tree with Phylip (5), using Neighbor-Joining, Maximum Likelihood and Parsimony algorithms (6). From the resulting tree, we were able to present a phage-lysin characterization network analysis taking into account the lysin aa sequence and the different phage classes (Family/Genus) and host species to study their evolutionary stories. Regarding the phage families, muramidases, amidases and peptidases are the largest type of lysins in Myoviridae, Podoviridae and Siphoviridae phages respectively. Grouped data will also be used to identify conserved domains among lysins of different phages which will play an important role in the annotation of the unidentified lytic cassette of sequenced phages

Insights into phage endolysins

(Bacterio)phages are viruses that specifically infect bacteria, and thus are harmless to humans, animals, and plants. They are the most abundant microorganisms on the planet (estimated to be 1031 on Earth) in a ratio of 10 times more than bacteria (1). Consequently, even rare phage-induced events are frequent at the global level. Therefore, they have a staggering ecological impact on the bacterial population and in the evolution of bacterial genomic structure upon virus-host interactions, acting as agents in the recycling of organic matter and presenting a valuable tool in molecular biology and epidemiology. Regarding the diversity of phages, they can have different types of replication mechanisms, morphologies, nucleic acid composition and genome sizes. Over the last decade improvements on phages genome sequencing and progresses in genomic research have revealed information on open reading frames of proteins of interest (2). Increasing interest has been given to phage (endo)lysins in molecular biology, biotechnology and medicine. Lysins are phage lytic enzymes that break down the peptidoglycan of the bacterial cell wall at the terminal stage of the phage reproduction cycle, in order to release the phage progeny with the consequent death of the bacterial cells (3). The number of phage genomes deposited in GenBank has been increasing exponentially in the last years. However, no effort has been made so far to understand the relation between lysins and their phage family and host species, presenting challenges in their annotation, comparative analysis, and representation. The almost 700 complete phage genomes deposited in the NCBI database were searched for the presence of lysins by making use of the Pfam (4) identified domains and BLAST comparison of putative or unidentified complete genome against known lysins. In approximately 5% of the phage genomes it was not possible to identify any lysin. The identified enzymes were used to construct a phylogenetic tree with Phylip (5), using Neighbor-Joining, Maximum Likelihood and Parsimony algorithms (6). From the resulting tree, we were able to present a phage-lysin characterization network analysis taking into account the lysin aminoacid sequence and the different phage classes (Family/Genus) and host species to study their evolutionary stories. Regarding the phage families, muramidases, amidases and peptidases are the largest type of lysins in Myoviridae, Podoviridae and Siphoviridae phages respectively. Grouped data will also be used to identify conserved domains among lysins of different phages which will play an important role in the annotation of the still unidentified lytic cassette of phages with sequenced genomes

Molecular aspects and comparative genomics of bacteriophage endolysins

Phages are recognized as the most abundant and diverse entities on the planet. Their diversity is predominantly determined by their dynamic adaptation capacities, when confronted with different selective pressures in an endless cycle of co-evolution with a widespread group of bacterial hosts. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that hinders their release into the environment and the opportunity to start a new infection cycle. Consequently, phages encode hydrolytic enzymes, called endolysins, to digest the peptidoglycan. In this work, we bring to light all phage endolysins found in completely sequenced double stranded nucleic acid phage genomes and uncover clues that explain the phage-endolysin-host ecology that led phages to recruit unique and specialized endolysins.This work was supported by a grant from the Portuguese Foundation for Science and Technology in the scope of the projects PTDC/AGR-ALI/100492/2008 and PTDC/AGR-ALI/121057/2010. Hugo Oliveira, Luis D. R. Melo, and Silvio B. Santos acknowledge the FCT grants SFRH/BD/63734/2009, SFRH/BD/66166/2009, and SFRH/BPD/75311/2010, respectively