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Analysis of the complete mitochondrial DNA from Anopheles funestus: An improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution
Abstract
Virtually no information regarding timing of deep lineage divergences within mosquito family (Culicidae) exists, which poses an important problem in the postgenomic era. To address this issue, the complete 15,354 bp mitochondrial genome of Anopheles funestus was assembled from both mtDNA and cDNA sequences generated from transcripts of the mtDNA-encoded protein and rRNA genes. Analysis of the transcript information allowed an improved genome annotation, revealing that the translation initiation codon for the cox1 gene is TCG, rather than atypical, longer codons proposed in several other insects. The 5'ends of nad1 and nad5 transcripts begin with TTG and GTG triplets, respectively, which apparently serve as the translation initiators for those genes. We used all the A. funestus mtDNA gene sequences and three other publicly available mosquito mtDNA genomes for the estimation of divergence time points within Culicidae. The maximum likelihood date estimates for the splits between Anopheles and Aedes (approximately 145-200 Mya), between Anopheles subgenera Cellia and Anopheles (approximately 90-106 Mya), and between lineages within subgenus Anopheles (approximately 70-85 Mya) inferred from protein-coding genes are roughly twice as high as the dates based on RNA gene sequences. Although existing evidence does not unequivocally favor one of the alternatives, fossil-based predictions of the age of the family Culicidae are in better agreement with dates inferred from protein-coding genes.
... The A+T content of An. sinensis mt genome is 78.34%, and the A+T contents of PCGs, tRNAs, rRNAs and CR are 76.85%, 78.59%, 81.46% and 93.58%, respectively ( Table 2). This result is similar to the universal feature presumed from earlier reported mosquito mt genomes [43][44][45][46][47][48][49][50][51] in that the CR has the highest A+T content, followed by rRNAs. For the 13 PCGs in the An. ...
... sinensis mt genome, the third codon position has a higher A+T content (94.24%), followed by the first codon position (69.46%) and the second codon position (66.84%). The results supports the data from other known mosquito mt genomes [43][44][45][46][47][48][49][50][51] in that the 3rd codon position has the highest A+T content, followed by the 1st codon position and 2nd codon position. AT-skew and GC-skew have also been widely used to measure the nucleotide compositional behaviors of mt genomes [42]. ...
... sinensis mt genome are 0.026 and -0.155, respectively ( Table 2). The AT-skew values are positive, and the GC-skew values are negative for all other mosquito mt genomes [43][44][45][46][47][48][49][50][51], which indicated overall mt genome preference for the use of A and C. The PCGs of the An. sinensis mt genome show an overall negative AT-skew (-0.144) and positive GC-skew (0.047). ...
Anopheles sinensis is a major malarial vector in China and Southeast Asia. The mitochondria is involved in many important biological functions. Nuclear mitochondrial DNA segments (NUMTs) are common in eukaryotic organisms, but their characteristics are poorly understood. We sequenced and analyzed the complete mitochondrial (mt) genome of An. sinensis. The mt genome is 15,418 bp long and contains 13 protein-coding genes (PCGs), two rRNAs, 22 tRNAs and a large non-coding region. Its gene arrangement is similar to previously published mosquito mt genomes. We identified and analyzed the NUMTs of 19 mosquito species with both nuclear genomes and mt genome sequences. The number, total length and density of NUMTs are significantly correlated with genome size. About half of NUMTs are short (< 200 bp), but larger genomes can house longer NUMTs. NUMTs may help explain genome size expansion in mosquitoes. The expansion due to mitochondrial insertion segments is variable in different insect groups. PCGs are transferred to nuclear genomes at a higher frequency in mosquitoes, but NUMT origination is more different than in mammals. Larger-sized nuclear genomes have longer mt genome sequences in both mosquitoes and mammals. The study provides a foundation for the functional research of mitochondrial genes in An. sinensis and helps us understand the characteristics and origin of NUMTs and the potential contribution to genome expansion.
... Five initiation codons were used in the present study, ATG (nad4, nad4L, cox2, cox3, and cytb), ATT (atp8, nad3, and nad6), TTG (atp6, nad1), GTG (nad5), and ATC (nad2). Similar to the previous proposal that ATT TAA and TCG triplet were the starts in the translation of the cox1 gene (Krzywinski et al. 2006), this structure also showed in the cox1 of Cx. vishnui. Additionally, those appearances of specific initiation codons were common in mosquitos, such as Anopheles gambiae (Beard et al. 1993). ...
... Except for the cox1 and cox2 genes in which incomplete termination codon T was used, the majority of the protein genes of Cx. tritaeniorhynchus and Cx. vishnui possessed the complete termination codon TAA, and this phenomenon has been extensively recorded because of posttranscriptional polyadenylation during which residual adenines could generate and added to originate TAA terminators in "cox" genes (Krzywinski et al. 2006;Krzywinska et al. 2016). The absence of TAA complete stop codon in the Cx. ...
Culex mosquitoes (Diptera: Culicidae) can transmit a variety of arthropod-borne viruses (arboviruses), causing human and animal diseases. Cx. vishnui, Cx. pseudovishnui, and Cx. tritaeniorhynchus are three representative species in Culex vishnui subgroup, which are widely distributed in southeast Asia, and they have been proved as the main vectors transmitting Japanese encephalitis virus (JEV) that could cause human infectious mosquito-borne disease across Asia. However, the epidemiology, biology, and even molecular information of those mosquitos remain poorly understood, and only the mitochondrial genome (mitogenome) of Cx. tritaeniorhynchus has been reported in these species. In the present study, we sequenced and annotated the complete mitogenome sequence of Cx. vishnui which was 15,587 bp in length, comprising 37 genes. Comparisons of nucleotide and amino acid sequences between Cx. vishnui and Cx. tritaeniorhynchus revealed that most genes within Culex vishnui subgroup were conserved, except atp8, nad1, atp6, and nad6, with differences of 0.4 (rrnS) — 15.1% (tRNAs) and 0 (nad4L) — 9.4% (atp8), respectively, interestingly suggesting the genes nad4L and rrnS were the most conserved but atp8 gene was the least. The results based on nucleotide diversity also supported a relatively uniform distribution of the intraspecific differences in Cx. vishnui and Cx. tritaeniorhynchus with only one highly pronounced peak of divergence centered at the control region. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes supported the previous taxonomic classification of the family Culicidae and the monophyly of tribes Aedini, Culicini, Mansoniini, and Sabethini. The present study revealed detailed information on the subgroup Culex vishnui, reanalyzed the relationships within the family Culicidae, provided better markers to identify and distinguish Culex species, and offered more markers for studying the molecular epidemiology, population genetics, and molecular phylogenetics of Cx. vishnui.
... evolved a preference for human hosts (White et al., 2011), as indicated by human adaptation to high malaria mortality occurring within the last 6,000 years (Flint et al., 1993;Tishkoff et al., 2001). Anthropophily in mosquitoes likely evolved several times independently, as major speciation events in mosquitoes occurred long before the emergence of hominins about 4.5 million years ago (Krzywinski et al., 2006;Neafsey et al., 2015;Prüfer et al., 2012). The two genera of Aedes and Anopheles diverged about 145 to 226 million years ago (Krzywinski et al., 2006;Reidenbach et al., 2009), with several species-specific expansions of the chemosensory gene repertoire observed to date (Arensburger et al., 2010;Bohbot et al., 2007;Neafsey et al., 2015). ...
... Anthropophily in mosquitoes likely evolved several times independently, as major speciation events in mosquitoes occurred long before the emergence of hominins about 4.5 million years ago (Krzywinski et al., 2006;Neafsey et al., 2015;Prüfer et al., 2012). The two genera of Aedes and Anopheles diverged about 145 to 226 million years ago (Krzywinski et al., 2006;Reidenbach et al., 2009), with several species-specific expansions of the chemosensory gene repertoire observed to date (Arensburger et al., 2010;Bohbot et al., 2007;Neafsey et al., 2015). As an example, the number of OR genes range from 77 in An. gambiae s.s. ...
... It has been estimated that the genus Anopheles originated in Western Gondwana [120] in what is now South America during the early Cretaceous period around 145 to 100 million years ago [121,122]. For the Albitarsis Complex, a monophyletic group, it has been estimated that it diverged approximately 39 MYA from its ancestor An. darlingi [123], following a latitudinal migration and subsequent diversification [121,124] and within the group the time divergence was estimated at 0.58-2.25 MYA [13]. ...
... It has been estimated that the genus Anopheles originated in Western Gondwana [120] in what is now South America during the early Cretaceous period around 145 to 100 million years ago [121,122]. For the Albitarsis Complex, a monophyletic group, it has been estimated that it diverged approximately 39 MYA from its ancestor An. darlingi [123], following a latitudinal migration and subsequent diversification [121,124] and within the group the time divergence was estimated at 0.58-2.25 MYA [13]. In accordance with the current natural geographical distribution registered for the Albitarsis Complex, we hypothesize that the origin of An. albitarsis s.l. was in the Precambrian Guayana Shield in southern Venezuela as the result of vertical migrations triggered by glacial/interglacial alternations followed by dispersal [125]. ...
Anopheles albitarsis F is a putative species belonging to the Albitarsis Complex, recognized by rDNA, mtDNA, partial white gene, and microsatellites sequences. It has been reported from the island of Trinidad, Venezuela and Colombia, and incriminated as a vector of malaria parasites in the latter. This study examined mitochondrially encoded cytochrome c oxidase I ( MT-CO1 ) sequences of An . albitarsis F from malaria-endemic areas in Colombia and Venezuela to understand its relations with other members of the Complex, revised and update the geographical distribution and bionomics of An . albitarsis F and explore hypotheses to explain its phylogenetic relationships and geographical expansion. Forty-five MT-CO1 sequences obtained in this study were analyzed to estimate genetic diversity and possible evolutionary relationships. Sequences generated 37 haplotypes clustered in a group where the genetic divergence of Venezuelan populations did not exceed 1.6% with respect to Colombian samples. Anopheles albitarsis F (π = 0.013) represented the most recent cluster located closer to An . albitarsis I (π = 0.009). Barcode gap was detected according to Albitarsis Complex lineages previously reported (threshold 0.014–0.021). Anopheles albitarsis F has a wide distribution in northern South America and might play an important role in the transmission dynamics of malaria due to its high expansion capacity. Future studies are required to establish the southern distribution of An . albitarsis F in Venezuela, and its occurrence in Guyana and Ecuador.
... The analysis of nucleotide diversity revealed that rrnL and rrnS were the most conserved genes, indicating that the ribosomal genes of the mosquito mitogenome do not provide sufficient information for the reconstruction of shallow phylogenetic relationships. Studies on the genera Drosophila (Cook, 2005) and Anopheles (Krzywinski et al., 2006) revealed negative selection patterns in mitochondrial ribosomal genes because some amino acid substitutions can influence secondary and tertiary protein structures. On the other hand, the highest values were found in the COX1, NAD2 and NAD6 genes. ...
... Our divergence dating indicates that Anophelinae diverged from Culicinae during the early Jurassic (197.5 Mya), which is consistent with other studies (Krzywinski et al., 2006;Hao et al., 2017;Tang et al., 2018). The basal split within Anophelinae was inferred in the early Cretaceous (∼147 Mya), in agreement with previous reports (Hao et al., 2017;Da Silva et al., 2020). ...
Understanding phylogenetic relationships within the family Culicidae informs mosquito evolution and may have public health implications as this family includes numerous species of medical and veterinary importance. We investigated the mitochondrial genomes of 102 mosquitoes, including six newly sequenced species, representing 21 genera with an emphasis on the Neotropical region. We estimated divergence times based on sequence data and three fossil calibration points, using Bayesian relaxed clock methods. Bayesian and maximum‐likelihood phylogenetic analyses based on the DNA sequences of 13 PCGs of the 102 species provided robust support for the monophyly of the subfamily Anophelinae and the tribes Aedini, Culicini, Mansoniini and Sabethini. Despite the current genera of Anophelinae being consistently recovered as monophyletic, relationships among them proved to be quite variable depending on the method used (concatenated or partitioned) and the number of taxa sampled. Molecular divergence time estimates revealed that the two mosquito subfamilies, Anophelinae and Culicinae, diverged in the early Jurassic (approximately 197.5 Mya). However, most major lineages of these groups arose after the Cretaceous, coincident with the emergence of angiosperms and the expansion of mammals and birds. The diversification and worldwide distribution of Culicidae may also be determined in part by geographic isolation as a result of continental drift during the Cretaceous.
... Use of Anopheles species allows for the resolution of CSB clusters that resemble those of Drosophila. Comparison of Anopheles with Culex and Aedes, separated by ~150 million years of evolutionary divergence [27], reveals uCSBs shared among these taxa. Although mosquitoes are considered to be Dipterans, uCSBs were conserved between mosquito species but not with ies. ...
... The CSB clusters resolved within the Anopheles species (data not shown) are similar to those detected using Dm as a reference sequence [30]. Phylogenic analysis has revealed the Anopheles species have diverged from ~48 My to ~30 My [31] while Aedes and Culex diversi ed from the Anopheles lineage in the Jurassic era (∼145-200 Mya) [27]) or even earlier. ...
Background Presence of ultra-conserved sequence elements in vertebrate enhancers suggest that transcription factor regulatory interactions are shared across phylogenetically diverse species. To date evidence for similarly conserved elements among evolutionarily distant insects such as flies, mosquitos, ants and bees, has been elusive. This study has taken advantage of the availability of the assembled genomic sequence of these insects to explore the presence of ultraconserved sequence elements in these phylogenetic groups.Results To investigate the integrity of fly regulatory sequences across ~100 million years of evolutionary divergence from the fruitfly Drosophila melanogaster , we compared Drosophila non-coding sequences to those of Ceratitis capitata , the Mediterranean fruit fly and Musca domestica , the domestic housefly. Using various alignment techniques, Blastn, Clustal, Blat, EvoPrinter and Needle, we show that many of the conserved sequence blocks (CSBs) that constitute Drosophila cis -regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca . We term the sequence elements shared among these species ultraconserved CSBs (uCSBs). The position of the uCSBs with respect to flanking genes is also conserved. Blastn alignments also detect putative cis -regulatory sequences shared among evolutionarily distant mosquitos Anopheles gambiae and Culex pipiens and Aedes aegypti. We have also identified conserved sequences shared among bee species. Side by side comparison of bee and ant EvoPrints identify uCSBs shared between the two taxa, as well as more poorly conserved CSBs in either one or the other taxon but not in both.Conclusions The results suggest that CSBs represent the point of interaction of multiple trans-regulators whose functions and interactions are conserved across divergent genera. Analysis of uCSBs in dipterans, mosquitos and bees will lead to a greater understanding of their evolutionary origin and the function of their conserved non-coding sequences.
... Use of Anopheles species allows for the resolution of CSB clusters that resemble those of Drosophila. Comparison of Anopheles with Culex and Aedes, separated by ~150 million years of evolutionary divergence ( Krzywinski et al. 2006), reveals uCSBs shared among these taxa. Although mosquitoes are considered to be Dipterans, uCSBs were conserved between mosquito species but not with flies. ...
... The CSB clusters resolved within the Anopheles species (data not shown) are similar to those detected using Dm as a reference sequence ( Brody et al, 2008). Phylogenic analysis has revealed the Anopheles species have diverged from ~48 My to ~30 My ( Kamali et al, 2014) while Aedes and Culex diversified from the Anopheles lineage in the Jurassic era (∼145-200 Mya; Krzywinski et al, 2006) or even earlier. ...
Presence of ultra-conserved sequence elements in vertebrate enhancers suggest that transcription factor regulatory interactions are shared across phylogenetically diverse species. To date evidence for similarly conserved elements among evolutionarily distant insects such as flies, mosquitos, ants and bees, has been elusive. This study has taken advantage of the availability of the assembled genomic sequence of these insects to explore the presence of ultraconserved sequence elements in these phylogenetic groups. To investigate the integrity of fly regulatory sequences across ~100 million years of evolutionary divergence from the fruitfly Drosophila melanogaster, we compared Drosophila non-coding sequences to those of Ceratitis capitata, the Mediterranean fruit fly and Musca domestica, the domestic housefly. Using various alignment techniques, Blastn, Clustal, Blat EvoPrinter and Needle, we show that many of the conserved sequence blocks (CSBs) that constitute Drosophila cis-regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca. We term the sequence elements shared among these species ultraconserved CSBs (uCSBs). The position of the uCSBs with respect to flanking genes is also conserved. The results suggest that CSBs represent the point of interaction of multiple trans-regulators whose functions and interactions are conserved across divergent genera. Blastn alignments also detect putative cis-regulatory sequences shared among evolutionarily distant mosquitos Anopheles gambiae and Culex pipiens and Aedes aegypti. We have also identified conserved sequences shared among bee species. Side by side comparison of bee and ant EvoPrints identify uCSBs shared between the two taxa, as well as more poorly conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in dipterans, mosquitos and bees will lead to a greater understanding of their evolutionary origin and the function of their conserved sequences.
... Counting nonsynonymous and synonymous substitutions separately in every codon along the branches of the tree, the likelihood of positive selection (v.1, where v = K A /K S ) is estimated allowing for heterogeneity in the mode and intensity of selection among codons. Considering that the time of divergence between the Culicinae and the Anophelinae exceeds 100 my [53], this analysis was aimed at evolutionary changes that occurred on a considerably ''deeper'' time scale than previous analyses, based on variation within and between populations of sibling species. ...
... At the longest time scale, spanning over 100 my of Culicidae evolution [53,61], PAML analysis detected strong positive selection on gambicin. At a single codon, nonsynonymous mutations occurred at a rate over 10 fold higher than the rate of synonymous mutations. ...
... Mya, in the late Jurassic (Fig. 8). This estimation is in reasonable agreement with the report by Krzywinski et al. 63 , who speculated the divergence time between Aedes (Stegomyia) and Anopheles was ~146 Mya based on mitochondrial DNA sequences. However, Zhou et al. 64 and Bertone et al. 65 pushed backward the split date to ~122 Mya, and the time estimated by Moreno M et al. 46 and Chen et al. 66 was ~190 Mya (Early Jurassic) and 217.5 Mya (Late Triassic), respectively. ...
... The split date (259.9 Mya) between Anopheles and Drosophila was used as the calibration 63,86 . ...
To better understand the phylogeny and evolution of mosquitoes, the complete mitochondrial genome (mitogenome) of Anopheles stephensi and An. dirus were sequenced and annotated, and a total of 50 mosquito mitogenomes were comparatively analyzed. The complete mitogenome of An. stephensi and An. dirus is 1,5371 bp and 1,5406 bp long, respectively. The main features of the 50 mosquito mitogenomes are conservative: 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, positive AT-skew and negative GC-skew. The gene order trnA-trnR in ancestral insects is rearranged. All tRNA genes have the typical clover leaf secondary structure but tRNASer. The control regions are highly variable in size. PCGs show signals of purifying selection, but evidence for positive selection in ND2, ND4 and ND6 is found. Bayesian and Maximum Likelihood phylogenetic analyses based on all PCG nucleotides produce an identical tree topology and strongly support the monophyly of subgenera Cellia, Anopheles, Keterszia and Nyssorhynchus, the sister relationship of the subgenera Nyssorhynchus and Keterszia, and Cellia and Anopheles. The most recent ancestor of the genus Anopheles and Culicini + Aedini exited ~145 Mya ago. This is the first comprehensive study of mosquito mitogenomes, which are effective for mosquito phylogeny at various taxonomic levels.
... They possess characteristics such as as single-parent inheritance and rapid evolution [10,11]. These characteristics have made mitochondrial genomes valuable for molecular marker screening [12,13], phylogenetic and evolutionary analyzes [14][15][16], and population genetics [17]. The mitochondrial genomes of common fungi are usually between 10 and over 100 kbp in length, and form a covalently closed circular structure [18,19]. ...
Backgroud
Paecilomyces variotii has important economic value in stimulating crop growth, biodegradation, and other aspects. Up to now, there are no research reports on its mitochondrial genome.
Methods and results
The mitochondrial genome of Paecilomyces variotii was determined with the next-generation sequencing method (Illumina, NovaSeq), and its characteristics were analyzed using various bioinformatics approaches. The length of complete mitochondrial genome sequence of P. variotii is 40,965 bp and consists of 14 protein-coding genes, 2 ribosomal RNA genes, 1 ribosomal protein S3 gene, 26 transport RNA genes. The results of phylogenetics analysis using Bayesian inference and Maximum likelihood methods showed that P. variotii belongs to the Eurotiales order in the Thermoascaceae family, and 9 genera within the Eurotiomycetes class were effectively distinguished with high support rates (bootstrap value > 92% and posterior probabilities > 99%). The analysis of synonymous substitution rates and nonsynonymous substitution rates indicated that the Ka/Ks values of the 14 PCGs in the mitochondrial genomes of the two orders in the Eurotiomycetes class ranged from 0 to 0.4333.
Conclusions
This study revealed the structural and sequence information characteristics of the mitochondrial genome of P. variotii, and the phylogenetic results strongly support its classification within the family Thermoascaceae, consistent with traditional morphological taxonomy studies. The 14 PCGs in the mitochondrial genomes of the two orders in the Eurotiomycetes class are subject to strong purifying (negative) selection. The results of this research provides an important molecular basis for the development of genomics, evolutionary genetics and molecular markers of P. variotii in the future.
... It would be interesting to determine which one, A. aegypti CLIPs A4, A14, or A4-A14, causes more A. aegypti PPO3 activation by A. aegypti CLIPB9x a (Ji et al., 2022). Functional tests of A4 and A14 orthologs in the two mosquitoes may inform us how conserved the cofactor role, if any, is after divergence of the mosquito lineages 145− 200 million years ago (Krzywinski et al., 2006). ...
... Analysis of mitochondrial DNA (mtDNA) is useful in reconstructing mosquito phylogenetic relationships, molecular evolution, and understanding their population history, i.e. time of taxon divergence [71]. For example, Michel et al. analyzed partial mitochondrial genes, NADH-ubiquinone oxidoreductase chain 5 protein (ND5) and Cytochrome c oxidase I (COI), from 11 countries across Africa, showing that An. funestus is genetically grouped into Eastern, Western, and Central populations, and detected two lineages I and II [59], defined as a group of mosquitoes that are ancestrally connected by using maternally inherited mitochondrial genes. ...
Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.
... Recent phylogenetic analysis of four markers (Cytb, COXII, aLAlb, and PRKCI) support monophyly of Muntiacinae (Elaphodus and Muntiacus) and estimates the origin of Muntiacinae at 7-8 million years ago (Gilbert et al., 2006). The complete mt genome provides a higher level of support than those based on individual or partial mt genes (Castro and Dowton, 2005;Krzywinski et al., 2006). Most taxonomic issues concern the genera of the family Cervidae: Cervus, Rucervus, and Rusa in the tribe Cervini, and Odocoileus, Mazama, and Pudu in the tribe Odocoileini. ...
... Aedes frucifer and Aedes taylori (Cook et al., 2005) DNA and cDNA sequences (Krzywinski et al., 2006). They studied the divergence time points within Culicidae. ...
Mosquitoes of the subfamilies Culicinae and Anophelinae of
the family Culicidae include some of the major vectors of diseases
like malaria, filariasis, yellow fever, dengue and dengue
hemorrhagic fever in human populations all over the world. Many
species of anopheline mosquitoes are the exclusive vectors of human
malaria in which some of the species dominate as the serious urban
and rural vectors. In the recent years the developments in DNA
based molecular cytogenetics have offered promising possibilities for
extension and future applications of genetic engineering based
control programmes. As a first step in integrated mosquito control
programmes, taxonomic and phylogenetic studies had been quite
productive and fairly successful in understanding the genetics of
vectorial capacity and insecticide resistance. Some of the molecular
level genomic studies involving in vitro amplification of DNA have
revealed that by using the technique of Polymerase chain reaction
(PCR), eukaryotic organisms have shown considerable nuclear DNA
polymorphisms which provide virtually unlimited opportunities for
establishing the exact taxonomic status of the species and studying
«
the mechanism of evolution in them. One of the most widely used
regions of the genome to infer genetic variations and phylogenetic
relationships is the rDNA gene cluster of tandemly repeated
multigene family.
... Mitochondrial genome sequences have been widely used as molecular markers for diverse evolutionary analyses because of their unique features, including coding content conservation, maternal inheritance and rapid evolution [32]. Insect mitochondrial genomes are usually small closed-circular molecules (15-20 kb) containing 13 proteincoding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a large non-coding element termed the A + T-rich or control region [21,33,34]. ...
The Chinese cordyceps, a parasitic Ophiocordyceps sinensis fungus–Thitarodes/Hepialus larva complex, is a valuable biological resource endemic to the Tibetan Plateau. Protection of the Plateau environment and huge market demand make it necessary to culture this complex in an artificial system. A method for the large-scale artificial rearing of the Thitarodes/Hepialus insect host has been established. However, the deterioration of the insect rearing population and low mummification of the infected larvae by the fungus constrain effective commercial cultivation. Hybridization of Thitarodes/Hepialus populations may be needed to overcome this problem. The species T. shambalaensis (GG♂ × GG♀) and an undescribed Thitarodes species (SD♂ × SD♀) were inbred or hybridized to evaluate the biological parameters, larval sensitivity to the fungal infection and mitochondrial genomes of the resulting populations. The two parental Thitarodes species exhibited significant differences in adult fresh weights and body lengths but not in pupal emergence rates. Hybridization of T. shambalaensis and Thitarodes sp. allowed producing a new generation. The SD♂ × GG♀ population showed a higher population trend index than the SD♂ × SD♀ population, implying increased population growth compared with the male parent. The sensitivity of the inbred larval populations to four fungal isolates of O. sinensis also differed. This provides possibilities to create Thitarodes/Hepialus populations with increased growth potential for the improved artificial production of the insect hosts. The mitochondrial genomes of GG♂ × GG♀, SD♂ × SD♀ and SD♂ × GG♀ were 15,612 bp, 15,389 bp and 15,496 bp in length, with an A + T content of 80.92%, 82.35% and 80.87%, respectively. The A + T-rich region contains 787 bp with two 114 bp repetitive sequences, 554 bp without repetitive sequences and 673 bp without repetitive sequences in GG♂ × GG♀, SD♂ × SD♀ and SD♂ × GG♀, respectively. The hybrid population (SD♂ × GG♀) was located in the same clade with GG♂ × GG♀, based on the phylogenetic tree constructed by 13 PCGs, implying the maternal inheritance of mitochondrial DNA.
... Therefore, a phylogenetic reconstruction based upon a single gene or a short DNA segment is highly likely to produce an incorrect tree topology (51). Several lines of evidence show that using the complete mitochondrial genome is a robust tool in order to gain complete phylogenetic relationships among taxa while using partial mitochondrial genes is not sufficient for this purpose (48)(49)(50)52). Considering that there was no research on the efficacy of the complete mitochondrial genomes in phylogenetic classification of Anopheles mosquitoes and the fact that some species of Anopheles mosquitoes are dangerous vectors of various diseases, including malaria, the present study evaluated the efficacy of the complete mtDNA genomes in proper separation and detecting the taxonomic and phylogenetic status of some of the species belonging to Anopheles genus. ...
Background: Among the blood-sucking insects, Anopheles mosquitoes have a very special position, because they transmit parasites of the genus Plasmodium, which cause malaria as one of the main vector-borne disease worldwide. The aim of this review study was to evaluate utility of complete mitochondrial genomes in phylogenetic classification of the species of Anopheles.
Methods: The complete mitochondrial genome sequences belonging to 28 species of the genus Anopheles (n=32) were downloaded from NCBI. The phylogenetic trees were constructed using the ML, NJ, ME, and Bayesian inference methods.
Results: In general, the results of the present survey revealed that the complete mitochondrial genomes act very accurately in recognition of the taxonomic and phylogenetic status of these species and provide a higher level of support than those based on individual or partial mitochondrial genes so that by using them, we can meticulously reconstruct and modify Anopheles classification.
Conclusion: Understanding the taxonomic position of Anopheles, can be a very effective step in better planning for controlling these malaria vectors in the world and will improve our knowledge of their evolutionary biology.
... Oxidor is fairly conserved between Cx. pipiens and Ae. aegypti, but based on phylogenetic analysis, Anopheles mosquitoes are evolutionarily more distant, a result which is consistent with reported speciation patterns within the family Culicidae (Krzywinski et al., 2006). ...
Throughout diapause in mosquitoes, stress resistance and subsequent prolonged lifespan are a few important features of diapause that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that oxidoreductin-like protein is involved with these diapause characteristics for overwintering survival. Expression of oxidor was more than two-fold higher in early stage diapausing females compared to their non-diapausing counterparts. Suppression of the gene that encodes oxidoreductin-like protein by RNAi significantly increased the proportion of degenerating follicles in early-stage adult diapausing females. Inhibition of oxidor also significantly reduced the survivability of diapausing females which indicates that this protein plays a key role in protecting multiple tissues during early diapause.
... This strongly suggests that transfer of steroids to females during mating is part of the "male 20E production" synapomorphy of Cellia mosquitoes. Our geographical mapping of nearly all contemporary mosquito species belonging to the Cellia subgenus (224 species) against the ones of Anopheles subgenus (184 species) (Fig. 2) suggests that the common ancestor of the Cellia subgenus diverged after separation of South America and Africa in agreement with previous observations 4,22 . This biogeographic calibration is consistent with divergence times obtained from our phylogenetic analysis placing the origin of steroid production by males of the Cellia subgenus around 84.1-69.2 ...
Human malaria, which remains a major public health problem, is transmitted by a subset of Anopheles mosquitoes belonging to only three out of eight subgenera: Anopheles, Cellia and Nyssorhynchus . Unlike almost every other insect species, it was shown that males of some Anopheles species produce and transfer steroid hormones to females during copulation and that this transfer mediates reproductive changes. Steroids are consequently seen as a potential target for malaria vector control. Here, we analysed the evolution of sexually-transferred steroids and their effects on female reproductive traits across Anopheles by using a set of 16 mosquito species (5 Anopheles , 8 Cellia , and 3 Nyssorhynchus ), including malaria vector and non-vector species. We show that male steroid production and transfer are specific to the Cellia subgenus and that there is no correlation between mating-induced effects in females and sexually-transferred steroids. In the light of our results, male steroid production, transfer and post-mating effects in females do not correlate with their ability to transmit human malaria, which overturns the suggestion from previous studies and suggests that manipulation of steroid-response pathways in the field should be considered with caution in order to benefit malaria vector control strategies.
... The gene shuffling is common in malaria mosquitoes [11], and these species are phylogenetically distant enough from each other [33,34] to have independently originated polymorphic inversions, which differ in chromosomal positions and size. The nonrandom presence of homologous genes within inversion 2Rb of An. gambiae and inversion 2Rh of An. funestus is especially interesting in the light of ecological adaptations associated with these inversions. ...
The malaria mosquito species of subgenus Cellia have rich inversion polymorphisms that correlate with environmental variables. Polymorphic inversions tend to cluster on the chromosomal arms 2R and 2L but not on X, 3R and 3L in Anopheles gambiae and homologous arms in other species. However, it is unknown whether polymorphic inversions on homologous chromosomal arms of distantly related species from subgenus Cellia nonrandomly share similar sets of genes. It is also unclear if the evolutionary breakage of inversion-poor chromosomal arms is under constraints.
To gain a better understanding of the arm-specific differences in the rates of genome rearrangements, we compared gene orders and established syntenic relationships among Anopheles gambiae, Anopheles funestus, and Anopheles stephensi. We provided evidence that polymorphic inversions on the 2R arms in these three species nonrandomly captured similar sets of genes. This nonrandom distribution of genes was not only a result of preservation of ancestral gene order but also an outcome of extensive reshuffling of gene orders that created new combinations of homologous genes within independently originated polymorphic inversions. The statistical analysis of distribution of conserved gene orders demonstrated that the autosomal arms differ in their tolerance to generating evolutionary breakpoints. The fastest evolving 2R autosomal arm was enriched with gene blocks conserved between only a pair of species. In contrast, all identified syntenic blocks were preserved on the slowly evolving 3R arm of An. gambiae and on the homologous arms of An. funestus and An. stephensi.
Our results suggest that natural selection favors specific gene combinations within polymorphic inversions when distant species are exposed to similar environmental pressures. This knowledge could be useful for the discovery of genes responsible for an association of inversion polymorphisms with phenotypic variations in multiple species. Our data support the chromosomal arm specificity in rates of gene order disruption during mosquito evolution. We conclude that the distribution of breakpoint regions is evolutionary conserved on slowly evolving arms and tends to be lineage-specific on rapidly evolving arms.
... The start codon of cox1 is full of controversial and designated as CGA in this study. The latter has been proved by a transcript experiment, with a clear cDNA sequence [23,24]. ...
The rice leafroller, Cnaphalocrocis medinalis, is one of the most important pests on rice and possesses striking flight ability. We have determined the nucleotide sequence of the 15,377 bp of a C. medinalis mitochondrial genome (mtDNA). The mtDNA encodes 37 genes and shows a unique lepidopteran CR-M-I-Q arrangement. Three possible substructures were detected in C. medinalis and some other lepidopteran insects’ control region. The findings might be relevant to the regulation of mtDNA replication and transcription. Phylogenetic relationships were reconstructed among 19 families in Lepidoptera so far. Cnaphalocrocis medinalis forms a reciprocal monophyletic group with Ostrinia in clade Crambidae instead of Pyralidae. The topology between Papilionoidea and other superfamilies showed an apparent contradiction with traditional Lepidopteran classification. As a well-known migratory insect, the molecular information contained in C. medinalis mtDNA may provide a further insight into the evolution of mitochondria genes and insect species, and may help to better understanding the energy metabolism of invertebrates.
... In general, the mitochondrial genome of most insects is a closed circular DNA molecule ranging in size from 14 to 20 kb, that encodes a common set of 37 genes (13 protein-coding genes, 22 tRNA genes, and two rRNA genes) and an A + T-rich region [18][19][20]. The mitochondrial genome is small-sized, with high coding content conservation, high evolutionary rates, maternal inheritance and rare recombination, and as such has been widely used for species identification and molecular evolutionary studies of Diptera [21][22][23][24][25][26]. Due to the rapid progress of sequencing technology, studies of Diptera mitogenomes have covered most families, especially medically significant taxa such as mosquitoes (Culicidae) [27][28][29], or economically important species from leafminers (Agromyzidae) and fruitflies (Tephritidae) [23,[30][31][32]. ...
... The exceptions were cox1 starting with CCG in all but P. taprobanensis, nad3 with TTG in P. taprobanensis, and nad1 with CTG in D. furumagiensis and TTG in D. fascipes, H. nigripes, P. taprobanensis, and S. anhuiensis. These anomalous start codons were proposed previously 13,30 and among them, CCG for cox1 13 and TTG for nad1 4,31 have been validated by transcript information. ...
The Trigonidiidae, a family of crickets, comprises 981 valid species with only one mitochondrial genome (mitogenome) sequenced to date. To explore mitogenome features of Trigonidiidae, six mitogenomes from its two subfamilies (Nemobiinae and Trigonidiinae) were determined. Two types of gene rearrangements involving a trnN-trnS1-trnE inversion and a trnV shuffling were shared by Trigonidiidae. A long intergenic spacer was observed between trnQ and trnM in Trigonidiinae (210−369 bp) and Nemobiinae (80–216 bp), which was capable of forming extensive stem-loop secondary structures in Trigonidiinae but not in Nemobiinae. The anticodon of trnS1 was TCT in Trigonidiinae, rather than GCT in Nemobiinae and other related subfamilies. There was no overlap between nad4 and nad4l in Dianemobius, as opposed to a conserved 7-bp overlap commonly found in insects. Furthermore, combined comparative analysis and transcript verification revealed that nad1 transcripts ended with a U, corresponding to the T immediately preceding a conserved motif GAGAC in the superfamily Grylloidea, plus poly-A tails. The resultant UAA served as a stop codon for species lacking full stop codons upstream of the motif. Our findings gain novel understanding of mitogenome structural diversity and provide insight into accurate mitogenome annotation.
... Compared with the ancestral gene arrangement of insects, T. eous possessed an inversion of the trnN-trnS1-trnE to trnE-trnS1-trnN, which was commonly found in all sequenced mitogenomes of the family Gryllidae. Most PCGs started with typical ATN codons (six with ATG, three with ATT, one with ATA, and one with ATC), whereas cox1 and nad1 started with non-canonical TCG and TTG, respectively, which were verified to act as start codons in transcript studies (Besansky et al. 2006;Stewart and Beckenbach 2009;Ma and Li 2018). Five PCGs ended with complete stop codons (TAA or TAG), while the others were proposed to utilize partial stop condons (T or TA) immediately preceeding a downstream gene. ...
The complete mitochondrial genome (mitogenome) of a field cricket Turanogryllus eous Bey-Bienko, 1956 was determined using next-generation sequencing. The mitogenome was 16,045 bp in length comprising 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region. Relative to the ancestral insect gene order, T. eous possessed an inversion of trnN-trnS1-trnE. The control region contained 3.4 tandem copies of a 194-bp sequence. Phylogenetic analysis supported that T. eous was sister to the clade comprising Teleogryllus and Velarifictorus. This study provides essential genetic information for genetic diversity analysis of T. eous.
... The split between the Culicidae family and other dipterans constitute one of the most ancient divergences of extant fly lineages [112]. The Culicidae family is comprised of the Culicinae and Anophelinae subfamilies, which separated between 150 and 200 mya [109,113]. Unfortunately, mosquito-borne diseases have coevolved with mosquitoes, exhibit highly efficacious infectivity, and may offer advantages to the mosquito [114,115], which makes control efforts exceptionally difficult [116]. Therefore, understanding these vectors, the environmental conditions they experience, and their associated pathogens, is of the upmost importance. ...
Diseases that are transmitted by mosquitoes are a tremendous health and socioeconomic burden with hundreds of millions of people being impacted by mosquito-borne illnesses annually. Many factors have been implicated and extensively studied in disease transmission dynamics, but knowledge regarding how dehydration impacts mosquito physiology, behavior, and resulting mosquito-borne disease transmission remain underdeveloped. The lapse in understanding on how mosquitoes respond to dehydration stress likely obscures our ability to effectively study mosquito physiology, behavior, and vectorial capabilities. The goal of this review is to develop a profile of factors underlying mosquito biology that are altered by dehydration and the implications that are related to disease transmission.
... For this reason, the phylogenetic relationships within the tribe Hippotragini in the tree in this study is slightly different than those based on seven complete mitochondrial genomes (specially in the case of the species belonging to the genus Hippotragus). The obtained topologies from complete mitochondrial genome provide a higher level of support than those based on individual or partial mitochondrial genes (Krzywinski et al., 2006) and probably the topology presented in this paper, is likely to be closer to reality. ...
In this survey, phylogenetic relationships within the tribe Hippotragini were re-evaluated using 132 gene sequences and ten complete mitogenomes. Based on the complete mitochondrial genomes, the average base composition of mtDNA sequences was 26.9 % T, 26.3 % C, 33.4 % A, and 13.4 % G, showing a strong AT bias (60.3 %). The phylogenetic trees were constructed using maximum likelihood and Bayesian inference methods, and were found to have very identical topologies. The results of this study revealed that this tribe is a strongly supported monophyletic group, and in all of the phylogenetic trees, except for COXI, Addax nasomaculatus is considered as a sister group for the monophyletic group of the genus Oryx. All of the species belonging to the genus Hippotragus constructed another monophyletic clade. Besides, within the monophyletic group of the genus Oryx, there are probably two distinct evolutionary pathways, i.e. O. gazella was located as a sister taxon of other species. Also, based on the whole mitogenomes, the extinct blaubok (H. leucophaeus), the endangered giant sable (H. niger variani), and the southern sable (H. niger niger), showed very close phylogenetic relationships and the roan antelope (H. equinus) is nested as their sister taxon.
... Mitochondrial DNA has a high mutation rate due to free oxygen radicals and less repair function than nuclear DNA (Richter et al. 1988). It is maternally inherited, evolves very quickly and is characterized by intraspecific polymorphisms (Krzywinski et al. 2006). Sequence differences are caused by mutation and not by recombination. ...
The Asian bush mosquito Aedes japonicus, endemic to East Asia, is one of the most expansive mosquito species in the world and has as yet established in 15 countries of Europe. Within Germany, the species has been spreading tremendously during the last years, and its four once geographically isolated populations were on the verge of merging in 2017. To reveal relationships and carry-over ways between the various populations, and thus, migration and displacement routes, the genetic make-up of Ae. japonicus from ten different locations throughout its German distribution area was investigated. For this purpose, a part of the mitochondrial DNA (nad4 gene) of collected specimens was sequenced and seven loci of short tandem repeats (microsatellites) were genotyped. When related to similar genetic studies carried out between 2012 and 2015, the results suggest that admixtures had since occurred, but no complete genetic mixture of populations had taken place. At the time of sampling for the present study, the western collection sites were still uniform in their genetic make-up; however, a carry-over of individuals from the southeastern to the northern and southwestern German populations was determined. Further introductions from abroad are possible. In summary, the genetic diversity of Ae. japonicus in Germany had grown considerably, thus increasing ecological variability and adaptability of the species. At this point (10 years after the first detection), it is not possible anymore to draw conclusions on the origins of the populations.
... This strongly suggests that transfer of steroids to females during mating is part of the "male 20E production" synapomorphy of Cellia mosquitoes. Our geographical mapping of nearly all contemporary mosquito species belonging to the Cellia subgenus (224 species) against the ones of Anopheles subgenus (184 species) (Fig. 2) suggests that the common ancestor of the Cellia subgenus diverged after separation of South America and Africa in agreement with previous observations 4,22 . This biogeographic calibration is consistent with divergence times obtained from our phylogenetic analysis placing the origin of steroid production by males of the Cellia subgenus around 84.1-69.2 ...
Human malaria, which remains a major public health problem, is transmitted by a subset of Anopheles mosquitoes belonging to only three out of eight subgenera: Anopheles, Cellia and Nyssorhynchus. Unlike almost every other insect species, males of some Anopheles species produce steroid hormones which are transferred to females during copulation to influence their reproduction. Steroids are consequently a potential target for malaria vector control. Here, we analysed the evolution of sexually-transferred steroids and their effects on female reproductive traits across Anopheles by using a set of 16 mosquito species (five Anopheles, eight Cellia, and three Nyssorhynchus), including malaria vector and non-vector species. We show that male steroid production and transfer are specific to the Cellia and therefore represent a synapomorphy of this subgenus. Furthermore, we show that mating-induced effects in females are variable across species and differences are not correlated with sexually-transferred steroids or with Anopheles ability to transmit human malaria. Overall, our findings highlight that Anopheles mosquitoes have evolved different reproductive strategies, independently of being a malaria vector or not.
... Bertone et al. (2008) used multiple nuclear genes to place the divergence time between Chaoboridae and Culicidae at 118 Mya. Using nuclear singlecopy genes and mitochondrial genome annotation, Krzywinski et al. (2006) proposed that Aedes and Anopheles separated 145-200 Mya, the split between Anopheles subgenera Cellia and Anopheles occurred at~90-106 Mya and the split between lineages within the subgenus Anopheles occurred around~70--85 Mya. A study by Moreno et al. (2010) used mtDNA genes to place the split between the Culicinae and Anophelinae at 190 Mya, with the radiation of Anopheles occurring 130 Mya. ...
A new genus and species of mosquito, Priscoculex burmanicus (Diptera: Culicidae: Anophelinae), are described from mid-Cretaceous Myanmar amber. Diagnostic characters for the holotype female are the presence of scales and setae on the wing veins, a setaceous, straight, proboscis that is subequal in length to the maxillary palpi, rounded margin of the scutellum, the third palpomere with a sensillum chaeticum, long setae on the proboscis and palpi, antennae longer than proboscis, absence of scales on the abdomen, lack of pulvilli and spiracular setae, truncate abdomen and oval cerci. The above-mentioned features are the basis for assigning Priscoculex as an early lineage of the Anophelinae that existed ~100–110 Mya. Discussion is presented on how the Anophelinae could have originated in Gondwana and colonized the interconnected lands before the continents separated.
www.zoobank.org/urn:lsid:zoobank.org:pub:C776F051-F345-4E15-9837-DF2D1AF22DF3www.zoobank.org/urn:lsid:zoobank.org:act:1601C12D-6CFC-49E9-8731-A011F98E6556www.zoobank.org/urn:lsid:zoobank.org:act:C5E4DBC1-CCE1-48F4-9A08-DAA6993BAA9D
... Based on our analyzes and on the principle of parsimony, we propose that Or2, Or9 and Or10 derive from two gene duplication events, one preceding the Anophelinae-Culicinae split that occurred 145-226 mya (Krzywinski et al., 2006;Reidenbach et al., 2009) and a second one that occurred in the Culicinae lineage ( Figure 4A). The most common recent ancestors of Or2 and Or10 were likely indole and skatole receptors since this function is still conserved in the two mosquito subfamilies (Bohbot et al., 2007). ...
Mosquitoes exhibit highly diverse and fast evolving odorant receptors (ORs). The indole-sensitive OR gene clade, comprised of Or2 and Or10 is a notable exception on account of its conservation in both mosquito subfamilies. This group of paralogous genes exhibits a complex developmental expression pattern in Aedes aegypti: AaegOr2 is expressed in both adults and larvae, AaegOr10 is adult-specific and a third member named AaegOr9 is larva-specific. OR2 and OR10 have been deorphanized and are selectively activated by indole and skatole, respectively. Using the two-electrode voltage clamp of Xenopus oocytes expressing Ae. aegypti ORs, we show that AaegOR9 is supersensitive and narrowly tuned to skatole. Our findings suggest that Ae. aegypti has evolved two distinct molecular strategies to detect skatole in aquatic and terrestrial environments, highlighting the central ecological roles of indolic compounds in the evolutionary and life histories of these insects.
... Studies have shown that IgG antibodies to SGE are more specific than IgM antibodies with less cross-reactivity to other mosquito vector species like Culex spp., but overall, Culex and Aedes appear to have species-specific saliva reactivity in terms of human antibody response given that they diverged over 55 million years ago [17,42]. There is little cross-reactivity in the Anopheles genera that diverged more than 150 million years ago from Aedes [43]. While these studies on markers of exposure laid the groundwork for anti-Aedes antibody identification and assay development, they have yet to contribute to our understanding of saliva-mediated pathogenesis of arboviral disease in humans. ...
Background
Mosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. When the infectious mosquitoes probe into human skin for a blood meal, they deposit saliva containing a myriad of pharmacologically active compounds, some of which alter the immune response and influence host receptivity to infection, and consequently, the establishment of the virus. Previous reports have highlighted the complexity of mosquito vector-derived factors and immunity in the success of infection. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered.
Methods/Design
We describe a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR.
Discussion
This study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia.
Trial registration
NCT03534245 registered on 23 May 2018.
... Protein coding genes were identified and annotated manually by sequence similarity to the previous reference genome (GenBank: DQ146364.1) as well as the orthologous sequences of other anopheline species 28,61,62 . Transfer RNA (tRNAs) were identified by their putative secondary structures using tRNAscan-SE 80 . ...
Anopheles funestus s.s. is a primary vector of malaria in sub-Saharan Africa. Despite its important role in human Plasmodium transmission, evolutionary history, genetic diversity, and population structure of An. funestus in southern and central Africa remains understudied. We deep sequenced, assembled, and annotated the complete mitochondrial genome of An. funestus s.s. for the first time, providing a foundation for further genetic research of this important malaria vector species. We further analyzed the complete mitochondrial genomes of 43 An. funestus s.s. from three sites in Zambia, Democratic Republic of the Congo, and Tanzania. From these 43 mitogenomes we identified 41 unique haplotypes that comprised 567 polymorphic sites. Bayesian phylogenetic reconstruction confirmed the co-existence of two highly divergent An. funestus maternal lineages, herein defined as lineages I and II, in Zambia and Tanzania. The estimated coalescence time of these two mitochondrial lineages is ~500,000 years ago (95% HPD 426,000-594,000 years ago) with subsequent independent diversification. Haplotype network and phylogenetic analysis revealed two major clusters within lineage I, and genetic relatedness of samples with deep branching in lineage II. At this time, data suggest that the lineages are partially sympatric. This study illustrates that accurate retrieval of full mitogenomes of Anopheles vectors enables fine-resolution studies of intraspecies genetic relationships, population differentiation, and demographic history. Further investigations on whether An. funestus mitochondrial lineages represent biologically meaningful populations and their potential implications for malaria vector control are warranted.
... The gene shuffling is common in malaria mosquitoes [11], and these species are phylogenetically distant enough from each other [33,34] to have independently originated polymorphic inversions, which differ in chromosomal positions and size. The nonrandom presence of homologous genes within inversion 2Rb of An. gambiae and inversion 2Rh of An. funestus is especially interesting in the light of ecological adaptations associated with these inversions. ...
Background:
Malaria mosquitoes have had a remarkable stability in the number of chromosomes in their karyotype (2n = 6) during 100 million years of evolution. Moreover, autosomal arms were assumed to maintain their integrity even if their associations with each other changed via whole-arm translocations. Here we use high-coverage comparative physical genome mapping of three Anopheles species to test the extent of evolutionary conservation of chromosomal arms in malaria mosquitoes.
Results:
In this study, we developed a physical genome map for Anopheles atroparvus, one of the dominant malaria vectors in Europe. Using fluorescence in situ hybridization (FISH) of DNA probes with the ovarian nurse cell polytene chromosomes and synteny comparison, we anchored 56 genomic scaffolds to the An. atroparvus chromosomes. The obtained physical map represents 89.6% of the An. atroparvus genome. This genome has the second highest mapping coverage among Anophelinae assemblies after An. albimanus, which has 98.2% of the genome assigned to its chromosomes. A comparison of the An. atroparvus, An. albimanus, and An. gambiae genomes identified partial-arm translocations between the autosomal arms that break down the integrity of chromosome elements in evolution affecting the structure of the genetic material in the pericentromeric regions. Unlike An. atroparvus and An. albimanus, all chromosome elements of An. gambiae are fully syntenic with chromosome elements of the putative ancestral Anopheles karyotype. We also detected nonrandom distribution of large conserved synteny blocks and confirmed a higher rate of inversion fixation in the X chromosome compared with autosomes.
Conclusions:
Our study demonstrates the power of physical mapping for understanding the genome evolution in malaria mosquitoes. The results indicate that syntenic relationships among chromosome elements of Anopheles species have not been fully preserved because of multiple partial-arm translocations.
... More specifically, just ten were classified as 1:1 orthologs and were predicted secreted proteins without transmembrane domains (Table 2). Overall, this suggests a potentially important physiological role for these proteins, as they have been conserved in two species that diverged more than 150 million years ago [45]. ...
The Aedes aegypti mosquito is the principal vector of arboviruses such as dengue, chikungunya, yellow fever, and Zika virus. These arboviruses are transmitted during adult female mosquito bloodfeeding. While these viruses must transverse the midgut to replicate, the blood meal must also reach the midgut to be digested, absorbed, or excreted, as aggregation of blood meal metabolites can be toxic to the female mosquito midgut. The midgut peritrophic matrix (PM), a semipermeable extracellular layer comprised of chitin fibrils, glycoproteins, and proteoglycans, is one such mechanism of protection for the mosquito midgut. However, this structure has not been characterized for adult female Ae. aegypti. We conducted a mass spectrometry based proteomic analysis to identify proteins that comprise or are associated with the adult female Ae. aegypti early midgut PM. Altogether, 474 unique proteins were identified, with 115 predicted as secreted. GO-term enrichment analysis revealed an abundance of serine-type proteases and several known and novel intestinal mucins. In addition, approximately 10% of the peptides identified corresponded to known salivary proteins, indicating Ae. aegypti mosquitoes extensively swallow their own salivary secretions. However, the physiological relevance of this remains unclear, and further studies are needed to determine PM proteins integral for midgut protection from blood meal derived toxicity and pathogen protection. Finally, we describe substantial discordance between previously described transcriptionally changes observed in the midgut in response to a bloodmeal and the presence of the corresponding protein in the PM. Data are available via ProteomeXchange with identifier PXD007627.
... Foster et al. [22] looked at relationships within Anopheles (Nyssorhynchus), and noted that recovery of known higher-level relationships benefited from more sequence data, and by extrapolation proposed using complete mitochondrial genomes for such problems in future. The mitochondrial genome is a rich source of information and has been used in several studies [23][24][25][26][27]. Analysis of complete mitochondrial genomes of Anopheles species has provided new evidence for species complexes and a new understanding of the phylogenetic relationships among them [27][28][29][30][31]. Similarly, promising results have been obtained for the classification of the Culex coronator species complex [26]. ...
Malaria is a vector-borne disease that is a great burden on the poorest and most marginalized communities of the tropical and subtropical world. Approximately 41 species of Anopheline mosquitoes can effectively spread species of Plasmodium parasites that cause human malaria. Proposing a natural classification for the subfamily Anophelinae has been a continuous effort, addressed using both morphology and DNA sequence data. The monophyly of the genus Anopheles, and phylogenetic placement of the genus Bironella, subgenera Kerteszia, Lophopodomyia and Stethomyia within the subfamily Anophelinae, remain in question. To understand the classification of Anophelinae, we inferred the phylogeny of all three genera (Anopheles, Bironella, Chagasia) and major subgenera by analysing the amino acid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culex species as outgroup taxa, supplemented with 23 mitogenomes from GenBank. Our analyses generally place genus Bironella within the genus Anopheles, which implies that the latter as it is currently defined is not monophyletic. With some inconsistencies, Bironella was placed within the major clade that includes Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia, which were found to be monophyletic groups within Anophelinae. Our findings provided robust evidence for elevating the monophyletic groupings Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia to genus level; genus Anopheles to include subgenera Anopheles, Baimaia, Cellia and Christya; Anopheles parvus to be placed into a new genus; Nyssorhynchus to be elevated to genus level; the genus Nyssorhynchus to include subgenera Myzorhynchella and Nyssorhynchus; Anopheles atacamensis and Anopheles pictipennis to be transferred from subgenus Nyssorhynchus to subgenus Myzorhynchella; and subgenus Nyssorhynchus to encompass the remaining species of Argyritarsis and Albimanus Sections.
... Mitochondrial genomes strictly follow maternal inheritance in structure and evolution, and contain abundant information for population genetic and phylogenetic studies [13][14][15][16][17][18][19][20][21]. There is no adequate mitogenome information available for An. ...
Background
Anopheles sinensis (Diptera: Culicidae) is a primary vector of Plasmodium vivax and Brugia malayi in most regions of China. In addition, its phylogenetic relationship with the cryptic species of the Hyrcanus Group is complex and remains unresolved. Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, of which mitochondrial genome data of An. sinensis is not available. Methods
An. sinensis samples was collected from Shandong, China, and identified by molecular marker. Genomic DNA was extracted, followed by the Illumina sequencing. Two complete mitochondrial genomes were assembled and annotated using the mitochondrial genome of An. gambiae as reference. The mitochondrial genomes sequences of the 28 known Anopheles species were aligned and reconstructed phylogenetic tree by Maximum Likelihood (ML) method. FindingsThe length of complete mitochondrial genomes of An. sinensis was 15,076 bp and 15,138 bp, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and an AT-rich control region. As in other insects, most mitochondrial genes are encoded on the J strand, except for ND5, ND4, ND4L, ND1, two rRNA and eight tRNA genes, which are encoded on the N strand. The bootstrap value was set as 1000 in ML analyses. The topologies restored phylogenetic affinity within subfamily Anophelinae. The ML tree showed four major clades, corresponding to the subgenera Cellia, Anopheles, Nyssorhynchus and Kerteszia of the genus Anopheles. Conclusions
The complete mitochondrial genomes of An. sinensis were obtained. The number, order and transcription direction of An. sinensis mitochondrial genes were the same as in other species of family Culicidae.
... The estimated divergence time within the subgenus Cellia (~41 mya) is consistent with divergence between the subgenera of Anopheles and Cellia (approximately 90-100 mya) as estimated by Krzywinski et al. (2006), following the separation of the Gondwana supercontinent from Laurasia around 100 mya during the late Cretaceous Period (Garros et al. 2005 (Morgan et al. 2009). ...
The evolutionary and taxonomic status is important for understanding speciation events and phylogenetic relationships between closely related vector and nonvector species. This information is useful for targeting important disease vector species groups for the development of novel genetic-based vector and pathogen control methods. In this study, different phylogenetic analyses were performed to reconstruct phylogenetic trees for the primary malaria vectors in Thailand based on sequence information of 4 DNA fragments from the nuclear and mitochondrial regions. The primary Anopheles species in the subgenus Cellia involved in malaria transmission in Thailand separate clearly into 3 distinct clades: the Leucosphyrus group, Minimus subgroup, and Maculatus group. The phylogenetic trees based on different reconstructed algorithms and different gene regions provided congruent phylogenetic status of the mosquito species studied. The phylogenetic relationships of malaria vector species examined followed similar patterns based on morphological characters. An estimate of the divergence time among the Anopheles species infers that they were present during the Eocene and Miocene periods (>41 million years ago). Congruent phylogenetic analysis of malaria vectors is presented with different algorithms and gene regions. The nuclear TOLL6 fragment appears useful for molecular phylogenetic, species DNA barcode, and Anopheles population genetic analyses.
Sex in honeybees, Apis mellifera , is genetically determined by heterozygous versus homo/hemizygous genotypes involving numerous alleles at the single complementary sex determination locus. The molecular mechanism of sex determination is however unknown because there are more than 4950 known possible allele combinations, but only two sexes in the species. We show how protein variants expressed from complementary sex determiner ( csd ) gene determine sex. In females, the amino acid differences between Csd variants at the potential-specifying domain (PSD) direct the selection of a conserved coiled-coil domain for binding and protein complexation. This recognition mechanism activates Csd proteins and, thus, the female pathway. In males, the absence of polymorphisms establishes other binding elements at PSD for binding and complexation of identical Csd proteins. This second recognition mechanism inactivates Csd proteins and commits male development via default pathway. Our results demonstrate that the recognition of different versus identical variants of a single protein is a mechanism to determine sex.
Mosquitoes are prolific vectors of human pathogens; a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.
Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of viruses and pathogens. Because mosquitoes are also highly diverse and globally widespread, their phylogeny has remained either little known or difficult to discern. Here, we used phylogenomic analysis of 709 orthologous nuclear gene sequences from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our tree supports an origin of mosquitoes in the early Triassic (~217 mya) with species diversification and host-use patterns within major lineages coinciding in earth history both with major geologic events and with the diversification of vertebrate classes.
Acronicta rumicis Linnaeus, 1758 is a notorious lepidopteran pest species that feed on economically impotant plants, including pears, apples and peaches. The outbreak of this pest can cause severe financial losses and threatens food security. Herein, we sequenced and annotated the entire mitochondrial genome (mitogenome) of A. rumicis. The A. rumicis mitogenome was 15,384 bp in size, containing 37 genes (13 protein coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs)) usually found in lepidopteran mitogenomes. The mitogenome’s nucleotide composition was highly biased towards using A and T, with positive AT skew and negative GC skew. All PCGs were initiated by typical ATN codons, except for the cox1, which was initiated by the CGA codon. The secondary structure of trnS1 lacked the common dihydrouridine (DHU) arm and replaced by a single loop. The seven bp overlapping region ‘ATGATAA’ located between atp6 and atp8, and the spacer region between trnS2 and nad1 contained an ‘ATACTAA’ motif, which are common in other sequenced lepidopterans. The A + T rich region was 370 bp and included a ‘ATAGA’ motif followed by a 19 bp poly-T stretch and a (AT)10 repeats. The phylogenetic analyses using the Maximum Likelihood (ML) and Bayesian Inference (BI) methods supported A. rumicis belonging to the subfamily Acronictinae. Furthermore, this study clarified a novel and monophyly relationship among Noctuidae with mitogenome data.
In mosquitoes, indolic compounds are detected by a group of olfactory indolergic Odorant Receptors (indolORs). The ancient origin of indole and 3-methylindole as chemical signals suggest that they may be detected by insects outside the Culicidae clade. To test this hypothesis, we have identified potential indolOR genes in brachyceran flies based on sequence homology. Because of the crucial roles of indolic compounds in oviposition and foraging, we have focused our attention on the housefly Musca domestica. Using a heterologous expression system, we have identified indolOR transcript expression in the female antennae, and have characterized MdomOR30a and MdomOR49b as 3-methylindole and indole receptors, respectively. We have identified a set of 92 putative indolOR genes encoded in the genomes of Culicoidea, Psychodidae and brachycera, described their phylogenetic relationships, and exon/intron structures. Further characterization of indolORs will impact our understanding of insect chemical ecology and will provide targets for the development of novel odor-based tools that can be integrated into existing vector surveillance and control programs.
In this study, the mitochondrial genome of Trifida elongate was sequenced, and comparative analysis of T. elongate and other 43 leafhoppers was performed based on the mitochondrial genome. The mitochondrial genome sequence length of T. elongate was 14,924 bp. It comprised 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, 22 transport RNA (tRNA) genes, and 1 non-coding control region. The control region is located between the rrnS and trnI genes, is characterized by two tandem repeats and three simple sequence repeats. Phylogenetic analysis showed that T. elongate is closely related with Bolanusoides shaanxiensis and Limassolla lingchuanensis (bootstrap value = 92% and posterior probabilities = 1). Analysis of synonymous and non-synonymous nucleotide substitutions showed that Ka/Ks value of the 13 protein-coding genes of 8 subfamily leafhoppers were less than 1 ranging from 0.0315 to 0.9928. atp8 had the highest Ka/Ks value whereas cox1 had the lowest Ka/Ks value. This study provides information on the structure and sequence characteristics of the mitochondrial genome of T. elongata. Typhlocybinae is clustered with (Cicadellinae+(Idiocerinae+(Mileewinae+(Nirvaninae+(Evacanthinae+Ledrinae))))).
The insect sex determination and the intimately linked dosage compensation pathways represent a challenging evolutionary puzzle that has been solved only in Drosophila melanogaster. Analyses of orthologs of the Drosophila genes identified in non-drosophilid taxa¹,² revealed that evolution of sex determination pathways is consistent with a bottom-up mode,³ where only the terminal genes within the pathway are well conserved. doublesex (dsx), occupying a bottom-most position and encoding sex-specific proteins orchestrating downstream sexual differentiation processes, is an ancient sex-determining gene present in all studied species.²,⁴,⁵ With the exception of lepidopterans, its female-specific splicing is known to be regulated by transformer (tra) and its co-factor transformer-2 (tra2).6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Here we show that in the African malaria mosquito Anopheles gambiae, a gene, which likely arose in the Anopheles lineage and which we call femaleless (fle), controls sex determination in females by regulating splicing of dsx and fruitless (fru; another terminal gene within a branch of the sex determination pathway). Moreover, fle represents a novel molecular link between the sex determination and dosage compensation pathways. It is necessary to suppress activation of dosage compensation in females, as demonstrated by the significant upregulation of the female X chromosome genes and a correlated female-specific lethality, but no negative effect on males, in response to fle knockdown. This unexpected property, combined with a high level of conservation in sequence and function in anopheline mosquitoes, makes fle an excellent target for genetic control of all major vectors of human malaria.
This study has taken advantage of the availability of the assembled genomic sequence of flies, mosquitos, ants and bees to explore the presence of ultraconserved sequence elements in these phylogenetic groups. We compared non-coding sequences found within and flanking Drosophila developmental genes to homologous sequences in Ceratitis capitata and Musca domestica . Many of the conserved sequence blocks (CSBs) that constitute Drosophila cis -regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca . Also conserved is the position but not necessarily the orientation of many of these ultraconserved CSBs (uCSBs) with respect to flanking genes. Using the mosquito EvoPrint algorithm, we have also identified uCSBs shared among distantly related mosquito species. Side by side comparison of bee and ant EvoPrints of selected developmental genes identify uCSBs shared between these two Hymenoptera, as well as less conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in these dipterans and Hymenoptera will lead to a greater understanding of their evolutionary origin and function of their conserved non-coding sequences and aid in discovery of core elements of enhancers.
This study applies the phylogenetic footprinting program EvoPrinter to detection of ultraconserved non-coding sequence elements in Diptera, including flies and mosquitos, and Hymenoptera, including ants and bees. EvoPrinter outputs an interspecies comparison as a single sequence in terms of the input reference sequence. Ultraconserved sequences flanking known developmental genes were detected in Ceratitis and Musca when compared with Drosophila species, in Aedes and Culex when compared with Anopheles , and between ants and bees. Our methods are useful in detecting and understanding the core evolutionarily hardened sequences required for gene regulation.
Whole mitogenome sequences (mtDNA) have been exploited for insect ecology studies, using them as molecular markers to reconstruct phylogenies, or to infer phylogeographic relationships and gene flow. Recent Anopheles phylogenomic studies have provided information regarding the time of deep lineage divergences within the genus. Here we report the complete 15,393 bp mtDNA sequences of Anopheles aquasalis, a Neotropical human malaria vector. When comparing its structure and base composition with other relevant and available anopheline mitogenomes, high similarity and conserved genomic features were observed. Furthermore, 22 mtDNA sequences comprising anopheline and Dipteran sibling species were analyzed to reconstruct phylogenies and estimate dates of divergence between taxa. Phylogenetic analysis using complete mtDNA sequences suggests that A. aquasalis diverged from the Anopheles albitarsis complex ~28 million years ago (MYA), and ~38 MYA from Anopheles darlingi. Bayesian analysis suggests that the most recent ancestor of Nyssorhynchus and Anopheles + Cellia was extant ~83 MYA, corroborating current estimates of ~79-100 MYA. Additional sampling and publication of African, Asian, and PLOS ONE | https://doi.org/10.1371/journal.pone.
BACKGROUND
Insecticides are still the main method of mosquito control, but mosquito resistance presents a large obstacle. The function of mitochondrial genes in the evolution of insecticide resistance is still poorly understood. Pyrethroid is the most commonly used insecticide, and Anopheles sinensis is an important malaria vector in China and Southeast Asia. In this study, we investigated the mitochondrial genes associated with pyrethroid resistance through their genetic and expression variation based on analyses of transcriptomes and 36 individuals with resequencing in three geographical populations in China.
RESULTS
The nucleotide diversity (Pi) in 18 resistant individuals was much lower than that in 18 susceptible individuals, which suggests that some sites experienced purifying selection subject to pyrethroid stress. Ka/Ks and amino acid analyses showed that ND4 experienced positive selection and had 23 amino acid mutations due to pyrethroid stress. These mutations might change the ND4 structure and function and thus alter the efficiency of the respiratory chain. ND5 was significantly upregulated, and ATP8 was significantly downregulated in these three pyrethroid resistant populations, which suggests that these two genes function in the production and maintenance of pyrethroid resistance. There are differences in mitochondrial genes involved in pyrethroid resistance among these three populations.
CONCLUSION
This is the first study to reveal the association of mitochondrial genes in the evolution of insecticide resistance through amino acid mutation and expression patterns and can help us further understand insecticide resistance mechanisms. © 2019 Society of Chemical Industry
The Anopheles mosquito Hyrcanus Group is widely distributed geographically across both Palearctic and Oriental regions and comprises 26 valid species. Although the species Anopheles sinensis Wiedemann (1828) is the most common in China and has a low potential vector rank, it has nevertheless long been thought to be an important natural malaria vector within the middle and lower reaches of the Yangtze River. A number of previous research studies have found evidence to support the occurrence of natural hybridization between An. sinensis and Anopheles kleini Rueda, 2005 (a competent malaria vector). We, therefore, collected a sample series of An. sinensis and morphologically similar species across China and undertook ribosomal and mitochondrial DNA analyses in order to assess genetic differentiation (Fst) and gene flow (Nm) amongst different groups. This enabled us to evaluate divergence times between morphologically similar species using the cytochrome oxidase I (COI) gene. The results of this study reveal significant genetic similarities between An. sinensis, An. kleini, and Anopheles belenrae Rueda, 2005 and therefore imply that correct molecular identifications will require additional molecular markers. As results also reveal the presence of gene flow between these three species, their taxonomic status will require further work. Data suggest that An. kleini is the most basal of the three species, while An. sinensis and An. belenrae share the closest genetic relationship.
Feeding upon vertebrate blood by mosquitoes permits transmission of diverse pathogens, including viruses, protozoa, and nematodes. Despite over a century of intensive study, no mosquito species is known to specialize on non-vertebrate hosts. Using molecular analyses and field observations, we provide the first evidence, to our knowledge, that a mosquito, Uranotaenia sapphirina, specializes on annelid hosts (earthworms and leeches) while its sympatric congener, Uranotaenia lowii, feeds only on anurans (frogs and toads). Our results demonstrate that Ur. sapphirina feeds on annelid hosts (100% of identified blood meals; n = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding on Sparganophilus worms and freshwater leeches. These findings indicate that adult mosquitoes utilize a much broader range of host taxa than previously recognized, with implications for epidemiology and the evolution of host use patterns in mosquitoes.
Haemosporidians are a diverse group of vector-borne parasitic protozoa that includes the agents of human
malaria; however, most of the described species are found in birds and reptiles. Although our understanding
of these parasites’ diversity has expanded by analyses of their mitochondrial genes, there is limited
information on these genes’ evolutionary rates. Here, 114 mitochondrial genomes (mtDNA) were studied
from species belonging to four genera: Leucocytozoon, Haemoproteus, Hepatocystis, and Plasmodium.
Contrary to previous assertions, the mtDNA is phylogenetically informative. The inferred phylogeny
showed that, like the genus Plasmodium, the Leucocytozoon and Haemoproteus genera are not
monophyletic groups. Although sensitive to the assumptions of the molecular dating method used, the
estimated times indicate that the diversification of the avian haemosporidian subgenera/genera took place
after the Cretaceous-Paleogene (K-Pg) boundary following the radiation of modern birds. Furthermore,
parasite clade differences in mtDNA substitution rates and strength of negative selection were detected.
These differences may affect the biological interpretation of mtDNA gene lineages used as a proxy to
species in ecological and parasitological investigations. Given that the mitochondria are critically important
in the parasite life cycle stages that take place in the vector and that the transmission of parasites belonging
to particular clades has been linked to specific insect families/subfamilies, this study suggests that
differences in vectors have affected the mode of evolution of haemosporidian mtDNA genes. The observed
patterns also suggest that the radiation of haemosporidian parasites may be the result of community-level
evolutionary processes between their vertebrate and invertebrate hosts.
Key Words ancestral polymorphism, coalescence theory, maximum likelihood, molecular clock, sequence saturation s Abstract Molecular clocks have profoundly influenced modern views on the timing of important events in evolutionary history. We review recent advances in es-timating divergence times from molecular data, emphasizing the continuum between processes at the phylogenetic and population genetic scales. On the phylogenetic scale, we address the complexities of DNA sequence evolution as they relate to estimating divergences, focusing on models of nucleotide substitution and problems associated with among-site and among-lineage rate variation. On the population genetic scale, we review advances in the incorporation of ancestral population processes into the estima-tion of divergence times between recently separated species. Throughout the review we emphasize new statistical methods and the importance of model testing during the process of divergence time estimation. 1 In memoriam: Joseph Slowinksi passed away in September 2001 as the result of a snake bite he received while conducting fieldwork in Myanmar. He will be dearly missed by his friends and colleagues.
Barmaculex antiquus new genus, new species, is new species, is describe from a single partially preserved adult female in burmese amber. The fossil has several plesiomorphic features, indicating that it is the sister group of all other fossil and extant Culicidae; a relatively short proboscis, the palpi extending beyond the apex of the proboscis, a clypeus with several setae, and the palpus without scales. Antennal and mouthpart structure suggest the female of this fossil species was a vertebrate blood feeder. The age of Burmese amber has been estimated as between Upper Albian to Turonian, 100-90 million years ago but the origins of the Culicidae are likely significantly older. The sister group of the Culicidae are the Chaoboridae, known as Jurassic fossils, and the Culicidae therefore must be equally as old. Although fossil adults of the two families may not be distinct at this early stage of evolution, the immatures would likely provide distinguishing features.
The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for tRNAs. The gene arrangement resembles those of arthropods much more than that of another mollusc, the bivalve Mytilus edulis. Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however, at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is unpaired in tRNA(ser)(AGN), which is typical of metazoan mtDNAs, and also in tRNA(ser)(UCN), a condition found previously only in nematode mtDNAs. There are two additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA(glu) and CO3 contains a 72-bp tract of alternating A and T.
A new genus and species of fossil mosquito (Diptera: Culicidae) is described from Canadian Cretaceous amber, thus providing the first undeniable record of this group from the Cretaceous Period. Paleoculicis minutus n.gen., n.sp. can be separated from extant culicids by features of the head, thorax, and abdomen. Paleoculicis has closer affinities to the Culicinae than to the Anophelinae or Toxorhynchitinae. If P. minutus fed on blood, a range of ve rtebrates (including dinosaurs) were potential hosts some 79 million years ago. A review of previous descriptions of fossil mosquitoes is presented. Many cannot be confidently assigned to the Culicidae, while others are extant species in copal. Only a minority of them can be regarded as true Culicidae, all of which are reported from Tertiary deposits.
The structure of the 5′-termini has been investigated in mitochondrial DNA- coded poly(A)-containing RNA from HeLa cells.
For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 μg/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose,
was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-
dimensional electrophoresis. No “cap” structures were detected under conditions where the presence of such structures in one
out of five to ten RNA molecules would have been recognized. It is, therefore, likely that “cap” structures are completely
absent in HeLa cell mitochondrial poly(A)-containing RNA.
The gene for subunit II of cytochrome oxidase (CoII) from bovine mitochondria has been cloned behind a T7 promoter and the corresponding mRNA synthesized in vitro. The RNA transcribed from this vector has a single nucleotide 5' to the start AUG and, thus, corresponds closely to the native mRNA. It binds to the small 28 S ribosomal subunit of bovine mitochondria but not to the large (39 S) subunit or to 55 S ribosomes. The binding occurs readily in the absence of auxiliary initiation factors or initiator tRNA. The complex formed appears to contain 1 mRNA/28 S subunit. The observed binding is specific for mRNA since neither tRNA nor ribosomal RNA can act as competitive inhibitors. The interaction of the mRNA with the 28 S subunit does not require an AUG codon near the 5' end and constructs containing 5' leaders of more than 100 nucleotides still bind efficiently. About 5% of the bound mRNA is protected from digestion by T1 RNase. The protected fragments do not arise from a specific region of the mRNA since they hybridize to several restriction fragments of the cloned CoII gene.
Genes for URF2, tRNAtrp, tRNAcys, tRNAtyr and cytochrome c oxidase subunit I (COI) have been identified within a sequenced segment of the Drosophila yakuba mtDNA molecule. The five genes are arranged in the order given. Transcription of the tRNAcys and tRNAtyr genes is in the same direction as replication, while transcription of the URF2, tRNAtrp and COI genes is in the opposite direction. A similar arrangement of these genes is found in mammalian mtDNA except that
in the latter, the tRNAala and tRNAasn genes are located between the tRNAtrp and tRNAcys genes. Also, a sequence found between the tRNAasn and tRNAcys genes in mammalian mtDNA, which is associated with the initiation of second strand DNA synthesis, is not found in this region
of the D. yakuba mtDNA molecule. As the D. yakuba COI gene lacks a standard translation initiation codon, we consider The possibility that the quadruplet ATAA may serve this
function. As in other D. yakuba mitochondrial polypeptide genes, AGA codons in the URF2 and COI genes do not correspond in position to arginine-specifying
codons in the equivalent genes of mouse and yeast mtDNAs, but do most frequently correspond to serine-specifying codons.
The neotropical Heliconius butterflies are famous examples of Müllerian mimicry, due to the diverse array of shared, brightly colored wing patterns that advertise the butterflies' unpalatability. The parallel geographical variation in these patterns within several widespread species has been invoked to support the controversial Pleistocene refugium hypothesis of tropical diversification. However, in no Heliconius species have either evolutionary rates or relationships among geographical races been explicitly examined. I present a phylogenetic hypothesis based on mitochondrial DNA sequences for 14 divergent races of Heliconius erato, which reveals that similar wing patterns have evolved rapidly and convergently within the species. There is a basal split between groups of races from east and west of the Andes, reflecting a vicariant separation at the base of the Pleistocene. Within each of these clades, sequence divergence is very low, and some haplotypes are shared between allopatric races with radically different wing patterns. The topology implies a simultaneous radiation of races in these two areas within the last 200,000 years. Ages for the clades are estimated by comparing sequence divergence to a plot of mitochondrial divergence in several arthropod taxa with independently dated divergence times. This plot is linear and suggests that mitochondrial DNA in arthropods evolves in a clocklike manner, at least initially, when sequence divergence is low.
The complete sequence (15,455 bp) of the mitochondrial DNA of the mosquito Anopheles quadrimaculatus species A is reported. This genome is compact and very A+T rich (77.4% A+T). It contains genes for 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement is the same as in Drosophila yakuba, except that the positions of two contiguous tRNAs are reversed and a third tRNA is transcribed from the complementary strand. Protein-coding genes, rRNAs, and most tRNAs were similar to D. yakuba. Two tRNAs had nonstandard secondary structures comparable with those of nematode mitochondrial tRNAs. The very small putative control region (625 bp) contains no sequence motifs similar to those used in vertebrates and other insects for initiation of transcription and replication.
We describe a program, tRNAscan-SE, which identifies 99–100% of transfer RNA genes in DNA sequence while giving less than
one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters
to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical
application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free
grammars. tRNAscan-SE searches at ∼30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as
selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudo-genes.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
The program MODELTEST uses log likelihood scores to establish the model of DNA evolution that best fits the data.
AVAILABILITY: The MODELTEST package, including the source code and some documentation is available at http://bioag.byu. edu/zoology/crandall_lab/modeltest.html.
Animal mitochondrial DNA is a small, extrachromosomal genome, typically ∼16 kb in size. With few exceptions, all animal mitochondrial
genomes contain the same 37 genes: two for rRNAs, 13 for proteins and 22 for tRNAs. The products of these genes, along with
RNAs and proteins imported from the cytoplasm, endow mitochondria with their own systems for DNA replication, transcription,
mRNA processing and translation of proteins. The study of these genomes as they function in mitochondrial systems—‘mitochondrial
genomics’—serves as a model for genome evolution. Furthermore, the comparison of animal mitochondrial gene arrangements has
become a very powerful means for inferring ancient evolutionary relationships, since rearrangements appear to be unique, generally
rare events that are unlikely to arise independently in separate evolutionary lineages. Complete mitochondrial gene arrangements
have been published for 58 chordate species and 29 non-chordate species, and partial arrangements for hundreds of other taxa.
This review compares and summarizes these gene arrangements and points out some of the questions that may be addressed by
comparing mitochondrial systems.
The entire mitochondrial gene order of the crustacean Pagurus longicarpus was determined by sequencing all but approximately 300 bp of the mitochondrial genome. We report the first major gene rearrangements
found in the clade including Crustacea and Insecta. At least eight mitochondrial gene rearrangements have dramatically altered
the gene order of the hermit crab P. longicarpus relative to the putatively ancestral crustacean gene order. These include two rearrangements of protein-coding genes, the
first reported for any nonchelicerate arthropod. Codon usage and amino acid sequences do not deviate substantially from those
reported for other crustaceans. Investigating the phylogenetic distribution of these eight rearrangements will add additional
characters to help resolve decapod phylogeny.
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We present the complete 15,455-nt mitochondrial DNA sequence of the springtail Tetrodontophora bielanensis (Arthropoda, Hexapoda, Collembola). The gene content is typical of most metazoans, with 13 protein-coding genes (PCGs), 2 genes encoding for ribosomal RNA subunits, and 22 tRNA genes. The nucleotide sequence shows the well-known A+T bias typical of insect mtDNA; its A+T content is lower (72.7%) than that observed in other insect species, but still higher than that in other arthropodan taxa. The bias appears to be uniform across the whole molecule, unlike other insect taxa, which show increased A+T content in the so-called A+T-rich region. However, the bias is slightly higher in the third codon positions of the PCGs (81.4%). Anomalous initiation codons have been observed in the nad2 and the cox1 genes. In the latter, the ATTTAA hexanucleotide is suggested to be involved in the initiation signaling. All tRNAs could be folded into the typical cloverleaf secondary structure, but the tRNA for cysteine appears to be missing the DHU arm. Long tandemly repeated regions (193 nt) were found in the A+T-rich region, which in turn was shown to have the possibility of forming a complex array of secondary structures. One of these structures encompassed the junction between the repeats. The A+T-rich region was also interesting in that it showed heteroplasmy in the number of repeats. Three haplotypes were found, possessing 2, 3, and 4 identical repeats, respectively. The order of protein coding and rRNA genes in the molecule was determined and was identical to that of all insects studied so far. However, two tRNA translocations were found which were unprecedented among Arthropoda. These involved the trnQ, which was found between the rrnS and the A+T-rich region, and the trnS(ucn), which was located between trnM and trnI. A preliminary phylogenetic analysis based on the amino acid sequence of the PCGs failed to find support for the monophyly of Hexapoda.
A phylogeny of the mosquito subfamily Anophelinae was inferred from fragments of two protein-coding nuclear genes, G6pd (462 bp) and white (801 bp), and from a combined data set (2,136 bp) that included a portion of the mitochondrial gene ND5 and the D2 region of the ribosomal 28S gene. Sixteen species from all three anopheline genera and six Anopheles subgenera were sampled, along with six species of other mosquitoes used as an outgroup. Each of four genes analyzed individually recovered the same well-supported clades; topological incongruence was limited to unsupported or poorly supported nodes. As assessed by the incongruence length difference test, most of the conflicting signal was contributed by third codon positions. Strong structural constraints, as observed in white and G6pd, apparently had little impact on phylogenetic inference. Compared with the other genes, white provided a superior source of phylogenetic information. However, white appears to have experienced accelerated rates of evolution in few lineages, the affinities of which are therefore suspect. In combined analyses, most of the inferred relationship were well-supported and in agreement with previous studies: monophyly of Anophelinae, basal position of Chagasia, monophyly of Anopheles subgenera, and subgenera Nyssorhynchus + Kerteszia as sister taxa. The results suggested also monophyletic origin of subgenera Cellia + Anopheles, and the white gene analysis supported genus Bironella as a sister taxon to Anopheles. The present data and other available evidence suggest a South American origin of Anophelinae, probably in the Mesozoic; a rapid diversification of Bironella and basal subgeneric lineages of Anopheles, potentially associated with the breakup of Gondwanaland; and a relatively recent and rapid dispersion of subgenus Anopheles.
In tropical Africa, Anopheles funestus is one of the three most important malaria vectors. We physically mapped 157 A. funestus complementary DNAs (cDNAs) to the polytene chromosomes of this species. Sequences of the cDNAs were mapped in silico to theA. gambiae genome as part of a comparative genomic study of synteny, gene order, and sequence conservation between A. funestus and A. gambiae. These species are in the same subgenus and diverged about as recently as humans and chimpanzees. Despite nearly perfect
preservation of synteny, we found substantial shuffling of gene order along corresponding chromosome arms. Since the divergence
of these species, at least 70 chromosomal inversions have been fixed, the highest rate of rearrangement of any eukaryote studied
to date. The high incidence of paracentric inversions and limited colinearity suggests that locating genes in one anopheline
species based on gene order in another may be limited to closely related taxa.
Organellar Genome Retrieval (OGRe) is a relational database of complete mitochondrial genome sequences for over 250 Metazoan
species. OGRe provides a resource for the comparative analysis of mitochondrial genomes at several levels. At the sequence
level, OGRe allows the retrieval of any selected set of mitochondrial genes from any selected set of species. Species are
classified using a taxonomic system that allows easy selection of related groups of species. Sequence alignments are also
available for some species. At the level of individual nucleotides, the system contains information on base frequencies and
codon usage frequencies that can be compared between organisms. At the level of whole genomes, OGRe provides several ways
of visualizing information on gene order. Diagrams illustrating the genome arrangement can be generated for any selected set
of species automatically from the information in the database. Searches can be done based on gene arrangement to find sets
of species that have the same order as one another. Diagrams for pairwise comparison of species can be produced that show
the positions of break-points in the gene order and use colour to highlight the sections of the genome that have moved. OGRe
is available from http://www.bioinf.man.ac.uk/ogre.
To help understand the mechanisms of gene rearrangement in the mitochondrial (mt) genomes of hemipteroid insects, we sequenced the mt genome of the plague thrips, Thrips imaginis (Thysanoptera). This genome is circular, 15,407 bp long, and has many unusual features, including (1) rRNA genes inverted and distant from one another, (2) an extra gene for tRNA-Ser, (3) a tRNA-Val lacking a D-arm, (4) two pseudo-tRNA genes, (5) duplicate control regions, and (6) translocations and/or inversions of 24 of the 37 genes. The mechanism of rRNA gene transcription in T. imaginis may be different from that of other arthropods since the two rRNA genes have inverted and are distant from one another. Further, the rRNA genes are not adjacent or even close to either of the two control regions. Tandem duplication and deletion is a plausible model for the evolution of duplicate control regions and for the gene translocations, but intramitochondrial recombination may account for the gene inversions in T. imaginis. All the 18 genes between control regions #1 and #2 have translocated and/or inverted, whereas only six of the 20 genes outside this region have translocated and/or inverted. Moreover, the extra tRNA gene and the two pseudo-tRNA genes are either in this region or immediately adjacent to one of the control regions. These observations suggest that tandem duplication and deletion may be facilitated by the duplicate control regions and may have occurred a number of times in the lineage leading to T. imaginis. T. imaginis shares two novel gene boundaries with a lepidopsocid species from another order of hemipteroid insects, the Psocoptera. The evidence available suggests that these shared gene boundaries evolved by convergence and thus are not informative for the interordinal phylogeny of hemipteroid insects. We discuss the potential of hemipteroid insects as a model system for studies of the evolution of animal mt genomes and outline some fundamental questions that may be addressed with this system.
The origin of New World monkeys (Infraorder Platyrrhini) has been an extensively debated issue. In this study, we analyzed mitochondrial genomes from Cebus (Platyrrhini), Homo, Hylobates, Pan, Pongo (Hominoids), Macaca, Papio (Cercopithecoids), and Tarsius (outgroup) to investigate this matter. Two distinct methodologies were employed on mitochondrial genes to estimate divergence times: the traditional likelihood ratio test performed in ML analyses of individual and concatenated gene sequences and the recent multigene Bayesian approach. Using the Cercopithecoid-Hominoid split as calibration point (25 MYA), our results show consistently that Platyrrhines split from Catarrhines at around 35 MYA. Although the main focus of the study is New World monkey origins, we have also estimated other primate divergence times: Homo-Pan at 5-7 MYA; Pongo-(Homo/Pan) at 13-16 MYA; Hylobates-(Pongo/Homo/Pan) at 15-19 MYA; and Macaca-Papio at 10-12 MYA. Our estimate for the origin of New World monkeys is in agreement with the hypothesis of a transatlantic journey from Africa to South America, as suggested by the fossil record.
The Afrotropical Funestus and the Oriental-African Minimus Groups of the Myzomyia Series of Anopheles subgenus Cellia are considered distinct only because of their geographical separation. For the first time, a phylogenetic study was done on six Oriental and four Afrotropical species based on the comparison of ribosomal (ITS2, D3) and mitochondrial (COI) nucleotide sequences. Both maximum parsimony (MP) and maximum likelihood (ML) analyses revealed that the groups form a monophyletic assemblage containing four clades. The inclusion of Afrotropical An. leesoni with the Oriental species was confirmed, whereas An. rivulorum, also an Afrotropical species, was placed in a basal position relative to the African and Oriental species. The biogeography of the Afrotropical and Oriental species was examined in relation to the phylogeny and estimates of divergence time. Divergence events correspond to periods of major tectonic movement as well as periods of great aridity or humidity.
The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for tRNAs. The gene arrangement resembles those of arthropods much more than that of another mollusc, the bivalve Mytilus edulis. Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however, at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is unpaired in tRNA(ser)(AGN), which is typical of metazoan mtDNAs, and also in tRNA(ser)(UCN), a condition found previously only in nematode mtDNAs. There are two additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA(glu) and CO3 contains a 72-bp tract of alternating A and T.
A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3′ end of the 28S β coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.
We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.
A phylogeny of the mosquito subfamily Anophelinae was inferred from fragments of two protein-coding nuclear genes, G6pd (462 bp) and white (801 bp), and from a combined data set (2,136 bp) that included a portion of the mitochondrial gene ND5 and the D2 region of the ribosomal 28S gene. Sixteen species from all three anopheline genera and six Anopheles subgenera were sampled, along with six species of other mosquitoes used as an outgroup. Each of four genes analyzed individually
recovered the same well-supported clades; topological incongruence was limited to unsupported or poorly supported nodes. As
assessed by the incongruence length difference test, most of the conflicting signal was contributed by third codon positions.
Strong structural constraints, as observed in white and G6pd, apparently had little impact on phylogenetic inference. Compared with the other genes, white provided a superior source of phylogenetic information. However, white appears to have experienced accelerated rates of evolution in few lineages, the affinities of which are therefore suspect.
In combined analyses, most of the inferred relationships were well-supported and in agreement with previous studies: monophyly
of Anophelinae, basal position of Chagasia, monophyly of Anopheles subgenera, and subgenera Nyssorhynchus + Kerteszia as sister taxa. The results suggested also monophyletic origin of subgenera Cellia + Anopheles, and the white gene analysis supported genus Bironella as a sister taxon to Anopheles. The present data and other available evidence suggest a South American origin of Anophelinae, probably in the Mesozoic;
a rapid diversification of Bironella and basal subgeneric lineages of Anopheles, potentially associated with the breakup of Gondwanaland; and a relatively recent and rapid dispersion of subgenus Anopheles.
Borkent, A.: A world catalogue of fossil and extant Corethrellidae and Chaoboridae (Diptera), with a listing of references to keys, bionomic information and descriptions of each known life stage. Ent. scand. 24: 1-24. Copenhagen, Denmark. April 1993. ISSN 0013-8711. A world list of species of fossil and extant Corethrellidae and Chaoboridae provides a catalogue of all valid names and their synonyms, original author, type-locality, type status and depository, distribution and the citation of authors who give the latest descriptions of the male adult, female adult, pupal, larval and egg stage. References to the most recent keys, descriptions, and bionomic information for each genus are also listed. A synopsis is given of the current state of systematic progress in each family. Nomenclatorial problems are also discussed. Sayomyia lanei Belkin, Heinemann & Page is a new junior synonym of Chaoborus braziliensis (Theobald), and Chaoborus annulatus Cook is recognized as a new junior synonym of C. festivus Dyar & Shannon. Corethrella kerrvillensis (Stone), Corethrella manaosensis (Lane & Cerqueira), and Chaoborus boliviensis (Lane & Heredia) are recognized as new combinations.
The generic relationships and higher classification of the family Culicidae are examined on the basis of a phylogenetic analysis. New and traditional morphological characters studied and compared throughout the Culicidae resulted in the acquisition of character data relative to the majority of species within each genus. Polymorphisms and morphological observations are discussed and additional information and illustrations are provided for the majority of characters and their character states. The analysis of seventy-three adult, pupal and fourth-instar larval characters coded for the thirty-eight currently recognized genera of mosquitoes resulted in relationships and groupings which differ significantly from traditional hypotheses. The analysis supports the monophyly of the subfamily Anophelinae and the tribes Culicini and Sabethini. The Anophelinae form the most basal clade of the family. The results indicate that Aedini is a paraphyletic assemblage with respect to the Mansoniini, each of which is monophyletic in itself. The Aedini + Mansoniini form a sister group to the Culicini + Sabethini, with the Aedini and the Culicini placed in ancestral relationships to the Mansoniini and the Sabethini, respectively. Based on the topography of generic relationships among more ‘generalized’ mosquitoes, the boundaries and relationships of the tribes Aedeomyiini, Uranotaeniini, Ficalbiini, Hodgesiini, Orthopodomyiini and Culisetini appear to be problematic. Relationships between genera of the tribe Aedini are generally poorly resolved due to a significant amount of polymorphism, especially within the genus Aedes as currently defined. There is no support for separate subfamily recognition for the genus Toxorhynchites, which is downgraded to tribal status as a result of the analysis. The results are discussed in relation to previous hypotheses based on subjective inference and cladistic analyses.
The ability to date the time of divergence between lineages using molecular data provides the opportunity to answer many important
questions in evolutionary biology. However, molecular dating techniques have previously been criticized for failing to adequately
account for variation in the rate of molecular evolution. We present a maximum-likelihood approach to estimating divergence
times that deals explicitly with the problem of rate variation. This method has many advantages over previous approaches including
the following: (1) a rate constancy test excludes data for which rate heterogeneity is detected; (2) date estimates are generated
with confidence intervals that allow the explicit testing of hypotheses regarding divergence times; and (3) a range of sequences
and fossil dates are used, removing the reliance on a single calculated calibration rate. We present tests of the accuracy
of our method, which show it to be robust to the effects of some modes of rate variation. In addition, we test the effect
of substitution model and length of sequence on the accuracy of the dating technique. We believe that the method presented
here offers solutions to many of the problems facing molecular dating and provides a platform for future improvements to such
analyses.
Higher-level relationships within Aedini, the largest tribe of Culicidae, are explored using morphological characters of eggs, fourth-instar larvae, pupae, and adult females and males. In total, 172 characters were examined for 119 exemplar species representing the existing 12 genera and 56 subgenera recognized within the tribe. The data for immature and adult stages were analysed separately and in combination using equal (EW) and implied weighting (IW). Since the classification of Aedini is based mainly on adult morphology, we first tested whether adult data alone would support the existing classification. Overall, the results of these analyses did not reflect the generic classification of the tribe. The tribe as a whole was portrayed as a polyphyletic assemblage of Aedes and Ochlerotatus within which eight (EW) or seven (IW) other genera were embedded. Strict consensus trees (SCTs) derived from analyses of the immature stages data were almost completely unresolved. Combining the adult and immature stages data resulted in fewer most parsimonious cladograms (MPCs) and a more resolved SCT than was found when either of the two data subsets was analysed separately. However, the recovered relationships were still unsatisfactory. Except for the additional recovery of Armigeres as a monophyletic genus, the groups recovered in the EW analysis of the combined data were those found in the EW analysis of adult data. The IW analysis of the total data yielded eight MPCs consisting of three sets of two mutually exclusive topologies that occurred in all possible combinations. We carefully studied the different hypotheses of character transformation responsible for each of the alternative patterns of relationship but were unable to select one of the eight MPCs as a preferred cladogram. Overall, the relationships within the SCT of the eight MPCs were a significant improvement over those found by equal weighting. Aedini and all existing genera except Ochlerotatus and Aedes were recovered as monophyletic. Ochlerotatus formed a polyphyletic assemblage basal to Aedes. This group included Haemagogus and Psorophora, and also Opifex in a sister-group relationship with Oc. (Not.) chathamicus. Aedes was polyphyletic relative to seven other genera, Armigeres, Ayurakitia, Eretmapodites, Heizmannia, Udaya, Verrallina and Zeugnomyia. With the exception of Ae. (Aedimorphus), Oc. (Finlaya), Oc. (Ochlerotatus) and Oc. (Protomacleaya), all subgenera with two or more species included in the analysis were recovered as monophyletic. Rather than leave the generic classification of Aedini in its current chaotic state, we decided a reasonable and conservative compromise classification would be to recognize as genera those groups that are 'weighting independent', i.e. those that are common to the results of both the EW and IW analyses of the total data. The SCT of these combined analyses resulted in a topology of 29 clades, each comprising between two and nine taxa, and 30 taxa (including Mansonia) in an unresolved basal polytomy. In addition to ten genera (Armigeres, Ayurakitia, Eretmapodites, Haemagogus, Heizmannia, Opifex, Psorophora, Udaya, Verrallina and Zeugnomyia), generic status is proposed for the following: (i) 32 existing subgenera of Aedes and Ochlerotatus, including nine monobasic subgenera within the basal polytomy, i.e. Ae. (Belkinius), Ae. (Fredwardsius), Ae. (Indusius), Ae. (Isoaedes), Ae. (Leptosomatomyia), Oc. (Abraedes), Oc. (Aztecaedes), Oc. (Gymnometopa) and Oc. (Kompia); (ii) three small subgenera within the basal polytomy that are undoubtedly monophyletic, i.e. Ae. (Huaedes), Ae. (Skusea) and Oc. (Levua), and (iii) another 20 subgenera that fall within the resolved part of the SCT, i.e. Ae. (Aedes), Ae. (Alanstonea), Ae. (Albuginosus), Ae. (Bothaella), Ae. (Christophersiomyia), Ae. (Diceromyia), Ae. (Edwardsaedes), Ae. (Lorrainea), Ae. (Neomelaniconion), Ae. (Paraedes), Ae. (Pseudarmigeres), Ae. (Scutomyia), Ae. (Stegomyia), Oc. (Geoskusea), Oc. (Halaedes), Oc. (Howardina), Oc. (Kenknightia), Oc. (Mucidus), Oc. (Rhinoskusea) and Oc. (Zavortinkius). A clade consisting of Oc. (Fin.) kochi, Oc. (Fin.) poicilius and relatives is raised to generic rank as Finlaya, and Downsiomyia Vargas is reinstated from synonymy with Finlaya as the generic name for the clade comprising Oc. (Fin.) leonis, Oc. (Fin.) niveus and their relatives. Three other species of Finlaya-Oc. (Fin.) chrysolineatus, Oc. (Fin.) geniculatus and Oc. (Fin.) macfarlanei- fall within the basal polytomy and are treated as Oc. (Finlaya) incertae sedis. Ochlerotatus (Ochlerotatus) is divided into three lineages, two of which, Oc. (Och.) atropalpus and Oc. (Och.) muelleri, are part of the basal polytomy. The remaining seven taxa of Oc. (Ochlerotatus) analysed, including the type species, form a reasonably well-supported group that is regarded as Ochlerotatus s.s. Ochlerotatus (Rusticoidus) is retained as a subgenus within Ochlerotatus s.s. Ochlerotatus (Nothoskusea) is recognized as a subgenus of Opifex based on two unique features that support their sister-group relationship. A new genus, Tanakaius gen. nov., is proposed for Oc. (Fin.) togoi and the related species Oc. (Fin.) savoryi. The taxonomic status and generic placement of all currently valid species of Aedini are listed in an appendix.
Intrastrand base pairings give ribosomal and other RNA molecules characteristic structures that are important for their function. In order to maintain these structures, a substitution at one paired site may have to be compensated for by an appropriate substitution at the complementary site. Thus paired sites do not evolve independently of one another. Most current methods for inferring phylogeny from molecular sequences assume that the sites are independent and will therefore give statistically unreliable and possibly erroneous results when used on structured RNA sequences. We analyze a new probabilistic model for the evolution of double-stranded RNA molecules that considers substitutions of the base pairs rather than of each of the bases independently. The new model, called the double-stranded model, was incorporated into the neighbor-joining distance and maximum likelihood methods. Computer simulations show that maximum likelihood is very robust to the violation of the assumption of the independence of sites. In contrast, the neighbor-joining method is sensitive to such violations: the double-stranded model can provide a significant increase in the chance of obtaining the correct tree topologies with neighbor joining when distances are large and the tree is difficult to obtain. The new model also leads to lower but more realistic estimates for the statistical confidence in the branch lengths and tree topologies.
Publisher Summary This chapter describes structural features and evolution of metazoan mitochondrial DNA (mtDNA) molecules. Throughout the evolution of metazoa, gene content of mitochondria-genomes is highly conserved, as has the close packing of genes. Most of the occasional sequence expansions that have occurred, by way of either repeated or noncoding unique sequences, are found in the control or putative control region, rather than being dispersed between genes. Of the 13 open reading frames recognized in the human mtDNA molecules, four (COI, COII, COIII, and Cyt b) are originally identified in regard to the proteins they encode, from similarities of their predicted amino acid sequences to known amino acid sequences of bovine proteins, and predicted amino acid sequences of yeast mt-protein genes. Among mtDNAs of vertebrates and higher invertebrates, there are genes that overlap. Some overlaps are among the 3′ ends of two genes that are encoded in opposite strands of the molecule. The extent of size reduction within metazoan mitochondrial-transfer RNA (mt-tRNA) gene sets strongly correlates with the degree to which the more variable secondary structure element-forming regions of mt-rRNA genes are lost.
A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3' end of the 28S beta coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.
The bovine mitochondrial gene products ND2 and ND4, components of NADH dehydrogenase, have been purified from a chloroform/methanol extract of mitochondrial membranes, and the human mitochondrial gene products ND2 and cytochrome b have been obtained by similar procedures. They have been identified by comparison of their amino-terminal protein sequences with those predicted from DNA sequences of bovine and human mitochondrial DNA. All of the proteins have methionine as their amino-terminal residue. In bovine ND2, this residue is encoded by the "universal" isoleucine codon AUA, and the sequences of human cytochrome b and bovine ND2 demonstrate that AUA also encodes methionine in the elongation step of mitochondrial protein synthesis. In human ND2, the amino-terminal methionine is encoded by AUU, which, as in the "universal" genetic code, is also used as an isoleucine codon in elongation. Thus, AUU has a dual coding function which is dependent upon its context.
The sequence of the 16,019 nucleotide-pair mitochondrial DNA (mtDNA) molecule of Drosophila yakuba is presented. This molecule contains the genes for two rRNAs, 22 tRNAs, six identified proteins [cytochrome b, cytochrome c oxidase subunits I, II, and III (COI-III), and ATPase subunits 6 and 8] and seven presumptive proteins (URF1-6 and URF4L). Replication originates within a region of 1077 nucleotides that is 92.8% A + T and lacks any open reading frame larger than 123 nucleotides. An equivalent to the sequence found in all mammalian mtCDNAs that is associated with initiation of second-strand DNA synthesis is not present in D. yakuba mtDNA. Introns are absent from D. yakuba mitochondrial genes and there are few (0-31) intergenic nucleotides. The genes found in D. yakuba and mammalian mtDNAs are the same, but there are differences in their arrangement and in the relative proportions of the complementary strands of the molecule that serve as templates for transcription. Although the D. yakuba small and large mitochondrial rRNA genes are exceptionally low in G and C and are shorter than any other metazoan rRNA genes reported, they can be folded into secondary structures remarkably similar to the secondary structures proposed for mammalian mitochondrial rRNAs. D. yakuba mitochondrial tRNA genes, like their mammalian counterparts, are more variable in sequence than nonorganelle tRNAs. In mitochondrial protein genes ATG, ATT, ATA, and in one case (COI) ATAA appear to be used as translation initiation codons. The only termination codon found in these genes is TAA. In the D. yakuba mitochondrial genetic code, AGA, ATA, and TGA specify serine, isoleucine, and tryptophan, respectively. Fifty-nine types of sense condon are used in the D. yakuba mitochondrial protein genes, but 93.8% of all codons end in A or T. Codon-anticodon interactions may include both G-A and C-A pairing in the wobble position. Evidence is summarized that supports the hypothesis that A and T nucleotides are favored at all locations in the D. yakuba mtDNA molecule where these nucleotides are compatible with function.
The sequence of a 4,869 base-pair fragment of Drosophila melanogaster mitochondrial DNA is presented. It contains genes for cytochrome oxidase subunits I, II and III, ATPase subunit 6 and six tRNAs together with two unassigned reading frames. The gene organization differs from that of mammalian mitochondrial DNAs. Evidence is provided for a genetic code in which AGA codes for serine and the quadruplet ATAA is used in initiation of translation.
A 3'-end proximal segment of most of the putative mRNAs encoded in the heavy strand of HeLa cell mtDNA has been partially sequences and aligned with the DNA sequence. In all cases, the 3'-end nucleotide of the individual mRNA coding sequences has been found to be immediately contiguous to a tRNA gene or another mRNA coding sequence. These and previous results indicate that the heavy (H) strand sequences coding for the rRNA, poly(A)-containing RNA and tRNA species form a continuum extending over almost the entire length of this strand. We propose that the H strand is transcribed into a single polycistronic RNA molecule, which is processed later into mature species by precise endonucleolytic cleavages which occur, in most cases, immediately before and after a tRNA sequence.
The 5'-end proximal sequences of all the putative mRNAs coded for by the heavy strand of HeLa cell mitochondrial DNA have been determined and aligned with the DNA sequence. All these mRNAs start directly at, or very near to, an AUG or AUA triplet, with the exception of one which starts at an AUU. The available evidence indicates that the terminal or subterminal AUGs and AUAs, and possibly also the terminal AUU, are initiator codons for the corresponding polypeptides. In most cases, the individual mRNA coding sequences are flanked on their 5' side by a tRNA gene, without any intervening nucleotide.
The nucleotide sequence of the regions flanking the A+T region of Drosophila melanogaster mitochondrial DNA (mtDNA) has been determined. Included are the genes encoding the transfer RNAs for valine, isoleucine, glutamine and methionine, the small ribosomal RNA and the 5'-coding sequences of the large ribosomal RNA and NADH dehydrogenase subunit II. This completes the nucleotide sequence of the D. melanogaster mitochondrial genome. The circular mtDNA of D. melanogaster varies in size among different populations largely due to length differences in the control region (Fauron & Wolstenholme, 1976; Fauron & Wolstenholme, 1980a, b); the mtDNA region we have sequenced, combined with those sequenced by others, yields a composite genome that is 19,517 bp in length as compared to 16,019 bp for the mtDNA of D. yakuba. D. melanogaster mtDNA exhibits an extreme bias in base composition; it comprises 82.2% deoxyadenylate and thymidylate residues as compared to 78.6% in D. yakuba mtDNA. All genes encoded in the mtDNA of both species are in identical locations and orientations. Nucleotide substitution analysis reveals that tRNA and rRNA genes evolve at less than half the rate of protein coding genes.
The entire 15,363 bp mitochondrial genome was cloned and sequenced from the mosquito Anopheles gambiae. With respect to the protein-coding genes, rRNA genes and the control region, the gene order was identical to that reported for other insects. There were significant differences, however, in the position and orientation of specific tRNA loci. The overall nucleotide composition was heavily biased towards adenine and thymine, which accounted for 77.6% of all nucleotides. Comparisons were made with the mitochondrial genomes of other insects on the basis genome size and organization, DNA and putative amino acid sequence data, nucleotide substitutions, codon usage and bias, and patterns of AT enrichment.
Relationships among the genus Anopheles and its many sibling species-groups are obscure despite the importance of anophelines as the vectors of human malaria. For the first time, the interrelationships and the origin of Australasian members of the subgenus Cellia are investigated by a cladistic analysis of sequence variation within the mitochondrial cytochrome oxidase subunit II gene. Estimated divergence times between many Australasian and Oriental taxa predate the mid Miocene collision of Australasia and Southeast Asia. Phylogenetic analysis suggests that two-way exchanges with Oriental mosquitoes rather than only immigration may have been a characteristic of anopheline paleobiogeography in Australasia. The Australasian fauna is mostly included in a large clade. The medically important Punctulatus Group is monophyletic and appears derived from Oriental stock. Populations within this group from as far apart as Australia and Vanuatu were in contact in the recent past (i.e., 0.35-2.44 mya), supporting dispersal rather than vicariance explanations. Some support for the monophyly of the Myzomyia, Neomyzomyia, and Pyretophorus Series was found. However, the subgenera Anopheles and Cellia and the Neocellia Series are paraphyletic, but branch support at these taxonomic levels was poor. The COII gene shows promise for questions concerning alpha taxonomy but appears to be of limited use for resolving deeper relationships within the Anopheles.
Mitochondria play a central role in cellular energy provision. The organelles contain their own genome with a modified genetic code. The mammalian mitochondrial genome is transmitted exclusively through the female germ line. The human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16569 bp and contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides. The mtDNA-encoded polypeptides are all subunits of enzyme complexes of the oxidative phosphorylation system. Mitochondria are not self-supporting entities but rely heavily for their functions on imported nuclear gene products. The basic mechanisms of mitochondrial gene expression have been solved. Cis-acting mtDNA sequences have been characterised by sequence comparisons, mapping studies and mutation analysis both in vitro and in patients harbouring mtDNA mutations. Characterisation of trans-acting factors has proven more difficult but several key enzymes involved in mtDNA replication, transcription and protein synthesis have now been biochemically identified and some have been cloned. These studies revealed that, although some factors may have an additional function elsewhere in the cell, most are unique to mitochondria. It is expected that cell cultures of patients with mitochondrial diseases will increasingly be used to address fundamental questions about mtDNA expression.
The sequence of the mitochondrial DNA (mtDNA) of the branchiopod crustacean Daphnia pulex has been completed. It is 15333bp with an A+T content of 62.3%, and contains the typical complement of 13 protein-coding, 22 transfer RNA (tRNA) and two ribosomal RNA (rRNA) genes. Comparison of this sequence with the sequences of the other eight completely sequenced arthropod mtDNAs showed that gene order and orientation are identical to that of Drosophila but different from Artemia due to the rearrangement of two tRNA genes. Nucleotide composition, codon usage, and amino acid composition are very similar in the crustaceans, but divergent from insects and chelicerates which show a much higher bias towards A+T. However, with few exceptions, the mitochondrial proteins of Daphnia are more similar to those of the dipteran insects (Drosophila and Anopheles) than to those of Artemia, at both the nucleotide and amino acid levels, suggesting that Artemia mtDNA is evolving at an accelerated rate. These results also show that sequence evolution and the evolution of nucleotide composition can be decoupled. Analysis of nucleotide substitution patterns in COII showed that there has been an unbiased acceleration of the overall substitution rate in Artemia. In contrast, the accelerated substitution rate in Apis is due partly to extreme A+T mutation pressure. Secondary structures are proposed for the Daphnia tRNAs and rRNAs. The tRNAs are similar to those of other arthropods but tend to have TPsiC arms that are only 4bp long. The rRNA secondary structures are similar to those proposed for insects except for the absence of a small number of helices in Daphnia. Phylogenetic analysis of second codon positions grouped Daphnia with Artemia, as expected, despite the latter's accelerated divergence rate. In contrast, the unusual pattern of mtDNA divergence in Apis led to a topology in which the holometabolous insects (Anopheles, Drosophila, Apis) appeared to be paraphyletic with respect to the hemimetabolous insect, Locusta, due to the early branching of Apis.
The utility of a mitochondrial DNA (mtDNA) fragment of about 1100 bp (including partial COI and COII sequences and tRNALeu) for evolutionary studies in Muscoidea is discussed. The species investigated are Scathophaga stercoraria, Microprosopa pallidicauda and Trichopalpus fraterna (family Scathophagidae), Musca domestica (Muscidae), Lasiomma seminitidum (Anthomyiidae) and Fannia armata (Fanniidae). Comparisons were made with published mtDNA sequences of Drosophila, Anopheles and three Calliphoridae species. The molecular phylogeny obtained here matches the classical morphological taxonomy reasonably well. This varies considerably, however, at different taxonomical levels. At a high taxonomic level, there is a clear separation between the Nematocera and the Brachycera, but the Calyptratae-Acalyptratae division is not always supported. At a lower taxonomic level, all species belonging to the same family are well grouped, but at an intermediate level, within the Calyptratae, it is impossible to clearly separate the Muscoidea and Calliphoridae, preventing a firm conclusion on the phylogenetic relationships among Muscoidea families. The entire COI sequence of S. stercoraria, as well as other mtDNA sequences (including the proximal portions of the COI gene, tRNATrp, tRNACys and tRNATyr genes) in Muscoidea species, are also presented and discussed.
A unified understanding of >390 Myr of insect evolution requires insight into their origin. Molecular clocks are widely applied for evolutionary dating, but clocks for the class Insecta have remained elusive. We now define a robust nucleotide and amino acid mitochondrial molecular clock encompassing five insect orders, including the Blattaria (cockroaches), Orthoptera (crickets and locusts), Hemiptera (true bugs), Diptera, and Lepidoptera (butterflies and moths). Calibration of the clock using one of the earliest, most extensive fossil records for insects (the early ancestors of extant Blattaria) was congruent with all available insect fossils, with biogeographic history, with the Cambrian explosion, and with independent dating estimates from Lepidopteran families. In addition, dates obtained from both nucleotide and amino acid clocks were congruent with each other.
Of particular interest to vector biology is the early date of the emergence of triatomine bugs (99.8–93.5 MYA), coincident with the formation of the South American continent during the breakup of Gondwanaland. More generally, we reveal the insects arising from a common ancestor with the Anostraca (fairy shrimps) at around the Silurian-Ordovician boundary (434.2–421.1 MYA) coinciding with the earliest plant megafossil. We explore Tilyard's theory proposing that the terrestrial transition of the aquatic arthropod ancestor to the insects is associated with a particular plant group (early vascular plants). The major output of the study is a comprehensive series of dates for deep-branching points within insect evolution that can act as calibration points for further dating studies within insect families and genera.
The century-old discovery of the role of Anopheles in human malaria transmission precipitated intense study of this genus at the alpha taxonomy level, but until recently little attention was focused on the systematics of this group. The application of molecular approaches to systematic problems ranging from subgeneric relationships to relationships at and below the species level is helping to address questions such as anopheline phylogenetics and biogeography, the nature of species boundaries, and the forces that have structured genetic variation within species. Current knowledge in these areas is reviewed, with an emphasis on the Anopheles gambiae model. The recent publication of the genome of this anopheline mosquito will have a profound impact on inquiries at all taxonomic levels, supplying better tools for estimating phylogeny and population structure in the short term, and ultimately allowing the identification of genes and/or regulatory networks underlying ecological differentiation, speciation, and vectorial capacity.
We report the complete mitochondrial DNA sequence of the spotted asparagus beetle, Crioceris duodecimpunctata. The genome complement, gene order, and nucleotide composition of this beetle's mitochondrial genome were found to be typical of those reported for other insects. Unusual features of this genome include the substitution of UCU for GCU as the anticodon for tRNA(Ser), an unusual TpsiC loop for the tRNA(Ile) gene, and the identification of a putative ATT start codon for cox1. The utility of complete mitochondrial genome data for phylogenetic inference of the insect orders was tested, and compared to that of cox1 and combined mitochondrial ribosomal DNA sequences. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the orders Diptera, Lepidoptera, and Coleoptera were consistently recovered. Monophyly of the Holometabola was also observed in some (though not all) analyses. The accumulation of complete mitochondrial sequences from a broader array of insect orders holds the promise of clarifying the early diversification of insects.
In view of the medical, sanitary and forensic importance of Chrysomya species, a knowledge of their nucleotide sequences would be useful for the molecular characterization of this genus, and would help in designing primers and in improving the molecular identification of Calliphoridae species. In this work, the mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae) was completely sequenced. The entire mitochondrial DNA (mtDNA) molecule was 15,837 bp long and was sequenced using the shotgun approach. The overall nucleotide composition was heavily biased towards As and Ts, which accounted for 76.7% of the whole genome. The cox1 gene had a serine as the start codon, while incomplete termination codons mediated by tRNA signals were found for cox2, nd4 and nd5. The C. chloropyga genes were in the same order and orientation as the mitochondrial genome of other dipteran species, except for the occurrence of a 123 bp region that included a complete duplication of tRNA(Ile) and a partial duplication of tRNA(Gln) genes. C. chloropyga is the first species of Diptera with 23 tRNA genes instead of the usual 22 already described. A phylogenetic analysis showed a split of Brachycera into Calyptratae and Acalyptratae subdivisions. The complete sequence of C. chloropyga mtDNA described here will be a useful source of sequence information for general molecular and evolutionary studies in Diptera.
Introduction to general state-space Markov chain theory. In W. R. Gilks, S. Richardson, and D. J. Spiegelhalter (Eds.), Markov Chain Monte Carlo in Practice, Chapter 4, pp. 59-74. London: Chapman and Hall. Yang, Z., R. Nielsen, N. Goldman, and A.-M. K. Pedersen (2000). Codon- substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155, 431-449. Yang, Z. and B. Rannala (1997). Bayesian phylogenetic inference using DNA sequences: A Markov chain Monte Carlo method. Molecular Biology and Evolution 1, 717-724. Yang, Z. H. (1993). Maximum-likelihood-estimation of phylogeny from DNA- sequences when substitution rates differ over sites. Molecular Biology and Evolution I0(6), 1396-1401. Zuckerkandl, E. (1987). On the molecular evolutionary clock. Journal of Molec- ular Evolution 26, 34-46. Zuckerkandl, E. and L. Pauling (1962). Molecular disease, evolution, and genic heterogeneity. In M. Kasha and B. Pullman (Eds.), Horizons in Biochem- istry, pp. 189-225. Ne