Genetic, haplotype and phylogenetic analysis of Ligula intestinalis by using mt-CO1 gene marker: ecological implications, climate change and eco-genetic diversity / Análise genética, haplótipo e filogenética de Ligula intestinalis usando marcador do gene mt-CO1: implicações ecológicas, mudanças climáticas e diversidade ecogenética

Ligula intestinalis is a cestode parasite that affects freshwater fish in different countries of the world. The current study aims to reveal the phylogenetic, genetic and haplotype diversity of mt-CO1 gene sequences sent to the NCBI database from different countries by using in-silico analysis. The 105 mt-CO1 (371 bp) gene sequences of L. intestinalis obtained from NCBI were used for bioinformatics analyses. Sequences were subjected to phylogenetic and haplotype analysis. As a result of the haplotype analysis of L. intestinalis, 38 haplotypes were obtained from 13 different countries. Hap24 constituted 44.76% of the obtained haplotype network. Changes in nucleotides between haplotypes occurred at 1-84 different points. China and Turkey have highest fixation index (Fst) values of 0.59761, while the lowest (-0.10526) was found between Russia and Turkey. This study provides a baseline for future studies on extensive scale on the epidemiology, ecological aspects, distribution pattern, transmission dynamics and population dispersion of L. intestinalis worldwide.

Ligula intestinalis é um parasita cestódeo que acomete peixes de água doce em diversos países do mundo. O presente estudo visa revelar a diversidade filogenética, genética e de haplótipos das sequências do gene mt-CO1 enviadas ao banco de dados do NCBI de diferentes países, por meio de análise in-silico. As sequências gênicas de 105 mt-CO1 (371 pb) de L. intestinalis obtidas do NCBI foram utilizadas para análises bioinformáticas. As sequências foram submetidas a análise filogenética e de haplótipos. Como resultado da análise de haplótipos de L. intestinalis, 38 haplótipos foram obtidos de 13 países diferentes. Hap24 constituiu 44,76% da rede de haplótipos obtida. Mudanças nos nucleotídeos entre os haplótipos ocorreram em 1-84 pontos diferentes. A China e a Turquia apresentam os maiores valores do índice de fixação (Fst), 0,59761, enquanto o menor (-0,10526) foi encontrado entre a Rússia e a Turquia. Este estudo fornece uma linha de base para futuros estudos em larga escala sobre epidemiologia, aspectos ecológicos, padrão de distribuição, dinâmica de transmissão e dispersão populacional de L. intestinalis em todo o mundo.

Population genetic structure and phenotypic diversity of Aspidodera raillieti (Nematoda: Heterakoidea), a parasite of Didelphini marsupials in Brazil's South and Southeast Atlantic Forest.

BACKGROUND: The population genetics of parasites may be influenced by host specificity, life cycle, host geographical range, evolutionary history, and host population structure. The nematode Aspidodera raillieti infects different marsupial and rodent hosts in the Nearctic and Neotropical regions, implying a gene flow among populations. However, niche diversification of the main hosts of A. raillieti in superimposed areas may provide conditions for population genetic structuring within this parasite species. We examined the genetic structuring of A. raillieti infecting three marsupial species co-occurring along the South and Southeast Brazilian Atlantic Forest, a hotspot of biodiversity. METHODS: We employed morphometric analyses and partial mitochondrial cytochrome c oxidase I gene sequences (MT-CO1) to characterize populations via phylogenetic and phylogeographic analyses. RESULTS: Among 175 A. raillieti specimens recovered from the marsupial hosts Didelphis aurita, D. albiventris, and Philander quica, we identified 99 MT-CO1 haplotypes forming four haplogroups and four clades in networks and phylogenetic trees, respectively. Clades I and II encompassed parasites of D. albiventris from the South region, clade III comprised parasites of D. aurita from the South and Southeast regions, and clade IV encompassed parasites of D. aurita and D. albiventris from the South and Southeast regions and parasites of P. quica from the South region. High genetic differentiation between clades, with a high fixation index and greater genetic variation in the analysis of molecular variance (AMOVA), indicated low gene flow between clades. Haplotypes shared among host species revealed a lack of host specificity. A significant correlation in the Mantel test suggested parasite isolation by distance, while there was no evidence of geographical structure between populations. Negative neutrality test values for clades III and IV suggested recent population expansion. Morphometric differentiation between A. raillieti specimens recovered from different host species, as well as from different localities, was more evident in males. CONCLUSION: The genetic structure of A. raillieti populations in the South and Southeast Atlantic Forest resulted from historical events rather than from current geographical distribution or host specificity. We also demonstrate morphometric variation associated with host species and localities, suggesting phenotypic plasticity to host attributes and to spatial variables.

DNA barcoding of the leaf-miner flies (Diptera, Agromyzidae) of Mitaraka, French Guiana.

Species level identification of Agromyzidae based on morphology is often challenging due to their small size and morphological homogeneity. DNA barcoding has been used regularly to assist with the identification of economically important species of Agromyzidae, but rarely as a tool for species delineation or identification in biodiversity surveys. The main objective of this study was to investigate whether DNA barcoding and the BIN (Barcoding Index) system could assist with species identification, species delineation, male/ female association, and diversity assessment of Agromyzidae material previously determined to morphospecies from Mitaraka, French Guiana. Amplification success was low, with sequences over 400 bp recovered for only 24 (48%) of the selected specimens. Sequences assigned to 17 morphospecies formed 16 distinct branches or clusters separated by very high (minimum of 10%) sequence divergence. Following the reassessment and subsequent reassignment of one specimen, congruence between morphology and DNA barcodes was high with a single instance of two morphospecies sharing identical sequences. While DNA barcoding did not assist with identification (none of our sequences matched those of named taxa in BOLD or GenBank), it did provide support for most of our morphospecies concepts, including male/female associations. The BIN system also provided access to information about the distribution and habitat preferences of several taxa. We conclude that DNA barcoding was a useful approach to study the species diversity of our samples but that much work remains to be done before it can be used as an identification tool for the Agromyzidae fauna of Mitaraka and the rest of the Neotropical region.

First report of the nematode Cruzia tentaculata using molluscs as natural intermediate hosts, based on morphology and genetic markers.

The life cycles of many parasitic nematodes include terrestrial gastropods as intermediate hosts. Over the past few decades, a number of cases of parasitism between molluscs and medically-important nematodes have been reported in Brazil, in particular, those involving the invasive giant African gastropod, Achatina fulica, and zoonoses caused by the nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, the etiological agents of neuroangiostrongyliasis and abdominal angiostrongyliasis, respectively. In the present study, larvae found infecting A. fulica, Latipes erinaceus, and Thaumastus taunaisii, from two localities in the Brazilian state of Rio de Janeiro were characterized using light and scanning electron microscopy, and sequences of the 18S rRNA and MT-CO1 genes. Genetic markers allowed to identify the larvae collected in the present study as Cruzia tentaculata, whose adults parasitize didelphid marsupials in the Americas. These findings indicate that both native and non-native gastropods may act as intermediate hosts and represent a previously unnoticed heteroxenous life cycle of C. tentaculata.

Revision and phylogeny of the genus Andocaeculus (Acari: Caeculidae) I: the A. weyrauchi species group.

Two new caeculid mite species, Andocaeculus beatrizrosso sp. nov. and Andocaeculus burmeisteri sp. nov., are described and A. weyrauchi (Franz, 1964) is redescribed based on material collected at the type locality. All post-larval stages are described for A. weyrauchi and Andocaeculus beatrizrosso sp. nov. and stochastic variation in the idiosomal and appendages chaetotaxy is considered. A clade of Andocaeculus containing the three species (the A. weyrauchi group) is established based on morphological characters, and confirmed with a Bayesian phylogenetic analysis of sequences from the CO1 marker. As result of the same analysis, the absence of the (st) pair of setae on leg II is proposed as a derived condition for the genus Andocaeculus, and the presence of the φ solenidion on leg IV is a derived condition for some Andocaeculus species of the A. weyrauchi species group.

Identification of phlebotomine sand flies (Diptera: Psychodidae) from leishmaniasis endemic areas in southeastern Mexico using DNA barcoding.

Leishmaniasis, a vector-borne disease transmitted to humans through the bite of phlebotomine sand flies, is of public health significance in southeastern Mexico. Active and continuous monitoring of vectors is an important aspect of disease control for the prediction of potential outbreaks. Thus, the correct identification of vectors is paramount in this regard. In this study, we employed DNA barcoding as a tool for identifying phlebotomine sand flies collected in localized cutaneous leishmaniasis endemic areas of Quintana Roo, Mexico. Specimens were collected using CDC light and Shannon traps as part of the Mexican Ministry of Health surveillance program. DNA extraction was carried out using a nondestructive protocol, and morphological identification based on taxonomic keys was conducted on slide-mounted specimens. Molecular taxonomic resolution using the 658-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was 100% congruent with the morphological identification. Seven species were identified: Lutzomyia cruciata (Coquillett 1907), Lutzomyia longipalpis (Lutz & Neiva 1912), Psathyromyia shannoni (Dyar 1929), Dampfomyia deleoni (Fairchild & Hertig 1947), Dampfomyia beltrani/steatopyga (Vargas & Díaz-Nájera 1951), Bichromomyia olmeca olmeca (Vargas & Díaz-Nájera, 1959), and Brumptomyia mesai (Sherlock 1962). Mean intraspecific divergence ranged from 0.12% to 1.22%, while interspecific distances ranged from 11.59% to 19.29%. Neighbor-joining (NJ) analysis using the Kimura 2-parameter model also showed specimens of the same species to be clustered together. The study provides the first cox1 sequences for three species of sand flies and indicates the utility of DNA barcoding for phlebotomine sand flies species identification in southeastern Mexico.

Leigh Syndrome Due to mtDNA Pathogenic Variants

Abstract Leigh syndrome is a devastating neurodegenerative disease, typically manifesting in infancy or early childhood. Hallmarks of the disease are symmetrical lesions in the basal ganglia or brain stem on MRI, and a clinical course with rapid deterioration of cognitive and motor functions. It is genetically heterogeneous, causative mutations have been disclosed in mitochondrial DNA and nuclear genes involved in the process of energy production in the mitochondria .We investigated the whole mitochondrial DNA in three Brazilian patients with LS, based on their clinical and biochemical data, with the aim to identify the disease-causing mutations. In two of the patients, with complex I deficiency, a novel heteroplasmic variant m.4142G>T (p.R279L) in MT-ND1 and a recurrent homoplasmic mutation m.10197G>A (p.A47T) in MT-ND3 were identified. In the remaining patient, with complex IV deficiency, a de novo heteroplasmic variant in MT-CO1 m.6547T>C (p.L215P) was found. The molecular investigation in mitochondrial diseases have shifted their focus from mitochondrial DNA to nuclear DNA, however, mtDNA protein-coding genes are one of the important genetic causes of mitochondrial disorders for Leigh syndrome. This study expands the molecular and clinical spectrum associated with this disease.

Proposed synonymy for Micropogonias altipinnis (Günther 1864), Micropogonias ectenes (Jordan & Gilbert 1882), and Micropogonias megalops (Gilbert 1890).

Within the Sciaenidae family, the genus Micropogonias is composed of three recognized species along the Pacific coast of Mexico: Micropogonias altipinnis, M. ectenes, and M. megalops. These species exhibit overlapping diagnostic characters, which make species identification difficult. This study ties morphological differences (meristic, morphometry of body, and otolith) with DNA sequences (CO1 and 16S fractions of mtDNA and 28S of nDNA) among Micropogonias species in the Pacific. Meristic analysis showed a latitudinal variation among the three species in the number of rays, the number of gill rakers, and length of the longest spine of the dorsal fin. Discriminant analysis of morphometric characters (body and otolith) showed three morphological entities (p < 0.001). However, the mean genetic divergences among the three species with partial sequences of mtDNA (CO1 and 16S), and nuclear (28S) were lower than those reported at the interspecific level (>2%). Genetic results suggest that the three species are one species and that the differences in meristics and morphometry could be the result of phenotypic plasticity or incipient speciation. In this sense, M. ectenes and M. megalops are proposed as junior synonyms of M. altipinnis.

Description and molecular characterization of a new species of tarantula, Pamphobeteus verdolaga, from Colombia (Araneae: Mygalomorphae: Theraphosidae)

A new species of Pamphobeteus Pocock, 1901, is described from the Colombian Andes, Medellín, Antioquia. The biochemistry and molecular characteristics of the venom of this new species (previously identified as Pamphobeteus aff. nigricolor ) has been already described. A detailed taxonomic study, comparing this species with holotypes and additional material of Pamphobeteus species, allowed us to recognize it as new, and to describe it here as Pamphobeteus verdolaga sp. nov. The male of P. verdolaga sp. nov. is distinguished by the palpal bulb with broad embolus, poorly developed apical keel (A), prolateral inferior keel (PI) and prolateral accessory keel (PAc) present but poorly developed, and retrolateral keel (R) of similar length as A. Females are distinguished by the morphology of spermatheca with a wide base and very short oval seminal receptacles, which are curved toward the center. This is the thirteenth species described in Pamphobeteus and the sixth species reported from Colombia. The species description is complemented by a molecular characterization of a partial CO1 sequence.(AU)

Description and molecular characterization of a new species of tarantula, Pamphobeteus verdolaga, from Colombia (Araneae: Mygalomorphae: Theraphosidae)

A new species of Pamphobeteus Pocock, 1901, is described from the Colombian Andes, Medellín, Antioquia. The biochemistry and molecular characteristics of the venom of this new species (previously identified as Pamphobeteus aff. nigricolor ) has been already described. A detailed taxonomic study, comparing this species with holotypes and additional material of Pamphobeteus species, allowed us to recognize it as new, and to describe it here as Pamphobeteus verdolaga sp. nov. The male of P. verdolaga sp. nov. is distinguished by the palpal bulb with broad embolus, poorly developed apical keel (A), prolateral inferior keel (PI) and prolateral accessory keel (PAc) present but poorly developed, and retrolateral keel (R) of similar length as A. Females are distinguished by the morphology of spermatheca with a wide base and very short oval seminal receptacles, which are curved toward the center. This is the thirteenth species described in Pamphobeteus and the sixth species reported from Colombia. The species description is complemented by a molecular characterization of a partial CO1 sequence.

Limited tolerance by insects to high temperatures across tropical elevational gradients and the implications of global warming for extinction.

The critical thermal maximum (CTmax), the temperature at which motor control is lost in animals, has the potential to determine if species will tolerate global warming. For insects, tolerance to high temperatures decreases with latitude, suggesting that similar patterns may exist along elevational gradients as well. This study explored how CTmax varies among species and populations of a group of diverse tropical insect herbivores, the rolled-leaf beetles, across both broad and narrow elevational gradients. Data from 6,948 field observations and 8,700 museum specimens were used to map the elevational distributions of rolled-leaf beetles on two mountains in Costa Rica. CTmax was determined for 1,252 individual beetles representing all populations across the gradients. Initial morphological identifications suggested a total of 26 species with populations at different elevations displaying contrasting upper thermal limits. However, compared with morphological identifications, DNA barcodes (cytochrome oxidase I) revealed significant cryptic species diversity. DNA barcodes identified 42 species and haplotypes across 11 species complexes. These 42 species displayed much narrower elevational distributions and values of CTmax than the 26 morphologically defined species. In general, species found at middle elevations and on mountaintops are less tolerant to high temperatures than species restricted to lowland habitats. Species with broad elevational distributions display high CTmax throughout their ranges. We found no significant phylogenetic signal in CTmax, geography, or elevational range. The narrow variance in CTmax values for most rolled-leaf beetles, especially high-elevation species, suggests that the risk of extinction of insects may be substantial under some projected rates of global warming.

Co1 DNA supports conspecificity of Geomyphiluspierai and G.barrerai (Coleoptera, Scarabaeidae, Aphodiinae) and is a good marker for their phylogeographic investigation in Mexican mountains.

Members of Geomyphilus are associated with rodent burrows, such as pocket gophers and prairie dogs. In Mexico, they are found in the mountains of the Mexican Volcanic Belt and in Sierra Madre Oriental. Our study aims to initiate the exploration of the dispersal modes of Geomyphiluspierai and Geomyphilusbarrerai from burrows of pocket gophers. In order to estimate the dispersal scale of the beetles, the utility of mitochondrial and nuclear molecular markers for studying the phylogeographic structure of this complex of species (Geomyphiluspierai and Geomyphilusbarrerai) was tested from 49 beetle individuals. High intraspecific and intra-mountain nucleotidic diversity was captured from this sample using Co1 mitochondrial sequences, whilst the ITS2 nuclear ribosomal sequence did not allow observing informative variation. Mitochondrial phylogenetic analysis revealed that the specific delineation between the two species under study was doubtful. In this preliminary study, Co1 was shown to be a good marker for elucidating dispersal routes of the burrowing rodent-associated beetles.

Phylogeny of the family Aglajidae (Pilsbry, 1895) (Heterobranchia: Cephalaspidea) inferred from mtDNA and nDNA.

The family Aglajidae includes several species of benthic, carnivorous cephalaspidean sea slugs, which generally lack a radula, have an internal shell, a posterior shield with short to moderate caudal lobes, and sensory cilia present on the head. The present study reports a phylogenetic analysis of the Aglajidae based on the mitochondrial genes 16S and CO1 and the nuclear gene H3, including 160 specimens of 54 species, that confirms the monophyly of Aglajidae as well as most taxonomically established genera, with some exceptions. Although support values are low for some clades, the analysis recovered the following clades within the Aglajidae: Odontoglaja, Nakamigawaia, and Melanochlamys. Chelidonura appears to be paraphyletic and the monophyly of a Chelidonura-Navanax-Aglaja clade is strongly supported in the Bayesian analysis, plus three of the four individual gene trees (COI, COI without 3rd codon positions, 16S and H3). However, the relatively low levels of support in the maximum likelihood analyses prevent us from proposing the synonymization of Navanax and Aglaja with Chelidonura. Melanochlamys is the sister clade of Chelidonura+Aglaja+Navanax. Odontoglaja is basal to the rest of the Aglajidae, confirming previous hypotheses on the loss of the radula in Aglajidae. Nakamigawaia and Melanochlamys are monophyletic, and should be maintained as valid. The monophyly of Philinopsis is strongly supported in the Bayesian analysis and in three of the four individual gene trees. Further research on this group is necessary to further affirm the monophyly of Chelidonura+Aglaja+Navanax and Philinopsis. Based on the results of this phylogenetic analysis, a reclassification of the taxonomy of Aglajidae is probably necessary. Additional genes should provide more information and probably fully resolve this situation. The present molecular study (including ABGD species delineation analyses) suggests the existence of previously undetected species complexes that require additional study to determine the extent of undocumented biodiversity.

New tools and insights to assist with the molecular identification of Simulium guianense s.l., main Onchocerca volvulus vector within the highland areas of the Amazonia onchocerciasis focus.

Following the success of the Onchocerciasis Elimination Programme for the Americas (OEPA), there is now just one Latin American onchocerciasis focus where onchocerciasis transmission is described as 'on-going:' the Amazonia Onchocerciasis focus. In the hyperendemic highland areas of the Amazonia focus, Simulium guianense s.l. Wise are the most important vectors of the disease. Populations of S. guianense s.l. are, however, known to vary in their cytogenetics and in a range of behaviours, including in their biting habits. In the hypoendemic lowland areas of the Amazonia focus, for example, S. guianense s.l. are generally regarded as zoophilic and consequently unimportant to disease transmission. Robust tools, to discriminate among various populations of S. guianense s.l. have, however, not yet been developed. In the work reported here, we have assessed the utility of a ribosomal DNA sequence fragment spanning the nuclear ribosomal ITS-1, ITS-2 and 5.8S sequence regions and a ∼850 nucleotide portion of the mitochondrial cytochrome oxidase gene (CO1) for species-level identification and for resolving the within species substructuring. We report here how we have generated 78 CO1 sequences from a rich set of both zoophilic and anthropophilic populations of S. guianense s.l. that were collected from eight sites that are broadly distributed across Brazil. Consistent with previous findings, our analysis supports the genetic isolation of Simulium litobranchium from S. guianense s.l. In contrast with previous findings, however, our results did not provide support for the divergence of the two species prior to the radiation of S. guianense s.l. In our analysis of the S. guianense s.l. ribosomal DNA sequence trace files we generated, we provide clear evidence of multiple within-specimen single nucleotide polymorphisms and indels suggesting that S. guianense s.l. ribosomal DNA is not a good target for conventional DNA barcoding. This is the first report of S. guianense s.l. within individual ribosomal DNA variation and thus the first evidence that the species is not subject to the normal effects of concerted evolution. Collectively, these data illustrate the need for diverse sampling in the development of robust molecular tools for vector identification and suggest that ribosomal DNA might be able to assist with resolving S. guianense s.l. species substructuring that C01 barcoding has hitherto failed to.

Evidence of geographical structuring in the Malaysian Snakehead, Channa striata based on partial segment of the CO1 gene.

Channa striata, locally known as "haruan", is economically important in fisheries and aquaculture industries in several Asian countries. DNA sequencing, based on a partial segment of the Cytochrome oxidase c subunit 1 (CO1) gene, was used to determine genetic variation in C. striata samples from four different populations on the west coast of Peninsular Malaysia. The highest nucleotide and haplotype diversities were observed in the Linggi population (π = 0.0067, h = 0.835), and the lowest in the Timah Tasoh population (π = 0.0008, h = 0.286). Apart from Kajang-Linggi, which was insignificant, F(ST) values were significant (p < 0.05) in all pairwise-population comparisons. Consequently, it is inferred that genetic structuring C. striata populations in this region was largely shaped by a common origin, with secondary influences from geographical factors and isolation.

Evidence of geographical structuring in the Malaysian Snakehead, Channa striata based on partial segment of the CO1 gene

Channa striata, locally known as "haruan", is economically important in fisheries and aquaculture industries in several Asian countries. DNA sequencing, based on a partial segment of the Cytochrome oxidase c subunit 1 (CO1) gene, was used to determine genetic variation in C. striata samples from four different populations on the west coast of Peninsular Malaysia. The highest nucleotide and haplotype diversities were observed in the Linggi population (π = 0.0067, h = 0.835), and the lowest in the Timah Tasoh population (π = 0.0008, h = 0.286). Apart from Kajang-Linggi, which was insignificant, F ST values were significant (p < 0.05) in all pairwise-population comparisons. Consequently, it is inferred that genetic structuring C. striata populations in this region was largely shaped by a common origin, with secondary influences from geographical factors and isolation.