Partial Mitochondrial Gene Research Articles

Phylogenetic relationships of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) with descriptions of two new species.

Ochoterenatrema is a small genus of lecithodendriid digeneans that includes six species parasitic in Neotropical bats in North and South America. Members of this genus are characterized by the presence of a pseudogonotyl formed by thickened tegument on the left side of the ventral sucker. We examined morphology of specimens belonging to five species of Ochoterenatrema from Brazil, Ecuador and the USA and describe two new Ochoterenatrema spp. (Ochoterenatrema sphaerula n. sp. and Ochoterenatrema gracilis n. sp.). These new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles and the body shape. We have provided the first DNA sequences for Ochoterenatrema. Newly generated partial nuclear 28S rDNA and mitochondrial cox1 gene sequences were used to differentiate among species and study the phylogenetic affinities of Ochoterenatrema spp., including one of the new species, O. gracilis n. sp., as well as Ochoterenatrema diminutum, Ochoterenatrema fraternum and Ochoterenatrema cf. labda. The phylogeny of the Microphalloidea based on 28S had well-supported topology, particularly at the family level. The Ochoterenatrema clade was strongly supported; however, the internal topology of the clade was weakly supported. Comparison of sequences revealed 0.4-1.3% interspecific divergence in 28S and 9.1-19.7% interspecific divergence in cox1 among Ochoterenatrema spp. We hypothesize that extremely diverse fauna of bats in South and Central America harbors multiple undescribed species of Ochoterenatrema. Several lecithodendriid genera from bats and other hosts are yet to be included in future molecular phylogenetic analyses to test the monophyly of this extremely diverse digenean family and analyze evolutionary affinities of its constituent taxa.

A tale about vipers’ tails: phylogeography of black-tailed rattlesnakes

The phylogenetic relationships among black-tailed rattlesnakes remain poorly understood and some authors indicated that the diversity of this group has been underestimated and additional analyses are required to clarify the biogeographic patterns throughout its distribution in Mexico. Therefore, the aim of this study was to elucidate the phylogenetic relationships among black-tailed rattlesnakes across their range, identifying relative divergence times among the main clades and reconstructing the biogeographical history of the group. Three partial mitochondrial genes (ND4, cytb and ATPase6) and one nuclear gene (RAG1) were sequenced to infer the phylogenetic relationships, through the maximum likelihood and Bayesian inference-based methods; demographic history reconstruction was investigated through Bayesian Skyline plot analysis and the ancestral area reconstruction was carried out considering a Bayesian framework. We found strong evidence that the black-tailed rattlesnakes’ group is composed of six clades, which is in agreement with subspecies previously reported. Divergence time estimation indicated that the origin of the C. molossus group could be traced to the middle of the Miocene (~7.71 Mya). Ancestral area reconstruction indicated that early divergence events occurred in Central Mexico, probably related to the geological dynamics of the Trans-Mexican Volcanic Belt. The lineage C. m. oaxacus is the basal member of the C. molossus group. Furthermore, the combination of geological events and changes in Quaternary vegetation may have contributed to the divergence of C. molossus clades. Our results suggest several clades within C. molossus complex could be potentially recognized as separate species.

Phylogenetic placement of the enigmatic Floodplain water snake, Lycodonomorphus obscuriventris FitzSimons, 1964

Lycodonomorphus is a genus of lamprophiid water snake endemic in Africa. Although widespread, abundant and presumably an important component of many aquatic and semi-aquatic food webs, these snakes are poorly understood taxonomically, particularly from a phylogenetic perspective. With only four of the nine species currently sequenced, this study attempts to improve our understanding of the evolutionary relationships within the genus through the phylogenetic placement of one of the most elusive species, Lycodonomorphus obscuriventris. Collected in the Ramsar declared Makuleke Wetlands in northern Kruger National Park (South Africa), the sample used in this study not only yielded the first DNA sequences for the taxon but also represented the most northerly South African record, bridging the gap between the southern and northern populations. The snake was sequenced for three partial mitochondrial genes (16S, Cyt-b, ND4) and one partial nuclear gene (c-mos) and phylogenetically placed, relative to the rest of the genus, using maximum likelihood (ML) and Bayesian inference (BI). Sequence divergences between sister taxa were also estimated using pairwise distance analysis. The concatenated phylogenetic reconstruction yielded similar topological structuring when compared to phylogenies from past articles, with both the ML and BI algorithms recovering strong support for L. obscuriventris as sister to a clade comprising of L. whytii + L. laevissimus + L. rufulus. The phylogenetic placement, albeit based on a single sample, challenges the original placement (morphological) of L. obscuriventris as sub-specific within L. whytii, suggesting that multiple species concepts should be considered when delineating species within this group.Conservation implications: Prior to the discovery of the new record, the global distribution of L. obscuriventris was characterised by two disjunct populations. The new record bridges the distribution gap between these two populations, rendering the distribution continuous. This bodes well for the species as there is likely no barrier to gene flow, thereby buffering the species from localised threats given the more expansive distribution. Furthermore, given that the specimen was sampled from the Kruger National Park, the species is likely to be well-protected as much of its distribution within South Africa seems to fall within protected areas.

Species identification and molecular analysis of terubuk fish from Bengkalis strait (Riau, Indonesia) using COI mitochondrial gene as a barcoding marker

Terubuk fish (Tenualosa spp.) commonly found in Bengkalis Strait is an important fishery commodity with a high economic value. However, the exploitation of the fish especially during spawning and hatching time due to the demand of the fish for consumption affects the decrease in numbers of the fish in Bengkalis Strait every year. Therefore, research on species identification and molecular analysis of the terubuk fish have to be investigated due to no genetic information can be used to make regulations and policies related to fish conservation. This research aimed to identify and examine the genetic polymorphism of terubuk fish in Bengkalis Strait using the partial COI mitochondrial gene. The method applied in this research was a PCR with primer FishF2 and FishR2. Genetic identification of the terubuk fish was analysed using nucleotide BLAST and Identification Engine through BOLD and genetic variation was evaluated using the DnaSP program. Genetic distance was examined using Kimura 2 parameter (K2P) model. The phylogenetic tree was constructed using Bayesian Inference through the BEAST program. The result revealed that all samples of terubuk fish were identified as Tenualosa macrura. The terubuk samples investigated in this study have 4 haplotypes with 6 variable sites dan 1 parsimony site. The haplotype diversity and nucleotide diversity were 0.714 and 0.00353 respectively with a genetic distance of 0-0.9% (mean= 0.4%). This finding is first reported and the genetic information gained in this study is expected to be implemented for terubuk fish conservation, especially in Bengkalis Strait.

Combining mitochondrial DNA and morphological data to delineate four new millipede species and provisional assignment to the genus

Hitherto, the millipede genus Apeuthes (family Pachybolidae, subfamily Trigoniulinae) was only known from three species described in Vietnam based on morphological characters. The present study uses two partial mitochondrial gene fragments (cytochrome c oxidase I (COI) and 16S ribosomal RNA) and morphology to define four new Apeuthes species from Malaysia, Thailand and Vietnam: A. fimbriatus, sp. nov., A. longeligulatus, sp. nov., A. pollex, sp. nov. and ?A. spininavis, sp. nov. The intraspecific COI sequence divergence of two Apeuthes species is 3–7% (mean: 5%) and the interspecific divergence of five species is 11–16% (mean: 13.7%). All members of the genus share unique male characters, viz the posterior gonopod telopodite with several dentate, serrate or tuberculate lamellae in a boat-like cavity or a boat-like cavity covered with spines. The delimitation of the four new species is supported by the congruence between mitochondrial DNA and morphological data. However, while the monophyly of Trigoniulinae is well supported, the relationships within this subfamily, and particularly among Apeuthes species, including the monophyly of Apeuthes, lack strong support. Therefore assignment of the four new species, and particularly of ?A. spininavis sp. nov., to the genus Apeuthes is tentative and awaits a comprehensive revision of the group.

First molecular description of Macracanthorhynchus hirudinaceus in wild boars from Italy with pathomorphological and epidemiological insights.

Macracanthorhynchus hirudinaceus is a zoonotic parasite affecting suids worldwide which are the definitive hosts for this helminth species. Macracanthorhynchus hirudinaceus is of significant economic and management concern due to its pathogenicity, causing intestinal obstruction and perforation in the definitive hosts. Current study is the preliminary investigation from Sardinia, Italy, reporting the pathomorphological findings and molecular characterization of M. hirudinaceus in the wild boars (Sus scrofa meridionalis). A total of 59 wild boars were examined showing acanthocephalan infection in 8 (13.6%) animals. In total, 49 parasites were collected with a mean intensity of 6.1. Comparatively higher infection levels were observed for males (16.7%) and young boars (14.3%); however, these epidemiological differences were statistically non-significant. Histopathological examination revealed the presence of a variable number of nodules (∼5 mm) in the intestine of M. hirudinaceus infested animals surrounded by a hyperemic-hemorrhagic halo. Several parasites were recovered from the intestinal lumen attached by the means of characteristic hooks showing necrosis in muscle layers. A moderate number of plump reactive fibroblasts and lesser numbers of fibrocytes were embedded with and at the borders of the inflammatory nodules in a moderate amount of homogeneous intensely eosinophilic fibrillary material rupturing the cell membrane. For molecular characterization, six isolated worms were amplified for the partial mitochondrial cox1 gene showing distinct interindividual variations. This first pathological and molecular description from southern Europe provided new knowledge about the diffusion of M. hirudinaceus in wild boars, furthering the research into the origin and transmission status of M. hirudinaceus in endemic localities.

A new species of freshwater crab of the genus Nanhaipotamon Bott, 1968 (Crustacea, Decapoda, Brachyura, Potamidae) from Longhai, Fujian Province, China.

A new species of freshwater crab of the genus Nanhaipotamon Bott, 1968 is described from Xiaye Village, Chengxiang Town, Longhai County, Zhangzhou City, Fujian Province, China. The new species is distinguished from congeners by the combination of characters of its carapace, third maxilliped, unequal chelipeds, triangular male abdomen and unique male first gonopod. Molecular evidence derived from partial mitochondrial 16S rRNA and COI genes also support the species as new.

Mitochondrial Differentiation and Biogeography of Rock Nuthatches

Rock Nuthatches comprise two morphologically similar species: Western Rock Nuthatch Sitta neumayer and Eastern Rock Nuthatch Sitta tephronota, with several subspecies within each species. These two species are closely related, and the two together are often considered an offshoot of the Sitta europaea group. The Western Rock Nuthatch is distributed across the Balkans to the Caucasus and Iran while the Eastern Rock Nuthatch is distributed across Eastern Anatolia, Iran, Afghanistan, Turkmenistan and Tajikistan. Using partial mitochondrial ND2 and ND3 gene sequences from populations throughout the distribution ranges and ecological niche modelling, we investigated the genetic structure and biogeographic history of these bird species. Two well-differentiated and supported clades in each species indicated a reciprocal monophyly with no gene flow between birds in the Zagros Mountains and other populations. Ecological niche modelling suggested population contraction in their potential habitat during the Last Glacial Maximum. The genetic differentiation in Zagros Mountains indicates that the post-glacial colonization of most of the Rock Nuthatch distribution range occurred from refugia located elsewhere than the Zagros Mountains. Given that Rock Nuthatches in the Zagros Mountains have long constituted an isolated population, there may be two phylogenetic species within each Rock Nuthatch species. Further genetic studies should be performed for taxonomic clarification.

Molecular Study of Echinococcus granulosus Cestodes in Ukraine and the First Genetic Identification of Echinococcus granulosus Sensu Stricto (G1 Genotype) in the Country.

Cystic echinococcosis is a globally distributed zoonotic disease of great medical and veterinary importance, which is caused by the cestode Echinococcus granulosus sensu lato. In Ukraine, two areas of the prominent circulation of the parasite are established, the southern steppe zone with sheep as the main transmitter, and the northern forest-steppe zone and Polissia, where pigs are mainly responsible for maintaining the E. granulosus transmission. Given that only a few studies have so far addressed the genetic diversity of the parasite in Ukraine, we have sequenced partial mitochondrial genes of cytochrome c oxidase 1 (789bp), NADH dehydrogenase 1 (602bp) and 12S rRNA (333-334bp) in pig metacestodes from the Sumy region (farms close to Sumy, northeastern Ukraine) and the Kyiv region (a farm in Bila Tserkva, central Ukraine). Four isolates from four pigs in the Sumy region were identified as E. canadensis (G7 genotype), the major E. granulosus s.l. species circulating in Eastern Europe, including the three microvariants (G7A, G7B, G7C). Three isolates from the two pigs in the Kyiv region were classified as E. granulosus s.s. (G1 genotype), including one microvariant (G1A). To our knowledge, this is the first genetic record of E. granulosus s.s. with the presumed highest infectivity and virulence among the E. granulosus s.l. species in Ukraine. The finding has implications for public health as local control programmes should take into consideration different development rate of this parasite in dogs and the greater risk of the species for human infection.

The Oriental Tiny Frog of the Genus <i>Microhyla</i> Tschudi, 1839 (Amphibia: Anura: Microhylidae) Revealed across Geographical Barriers of the Wallace Line

The frog genus Microhyla was considered as the South, East, and Southeast Asian frog species. Microhyla orientalis was described in 2013, distributed in Java and Bali, Indonesia. Thenceforth, it was known as the easternmost distribution of this genus within the oriental region, but recently this species was recorded from the Timor Island and Sulawesi on the Wallace regions. We applied molecular analysis to evaluate the taxonomic status and the origin of the Wallacean population. Phylogenetic analysis using the partial 16S mitochondrial gene demonstrated that the Java, Timor and Sulawesi populations were not significantly different from the Bali population. This Wallacean population of M. orientalis was originated from Java and possibly it is accidentally distributed by humans through the expansion of agricultural activity.

Utility of Complete Mitochondrial Genomes in Phylogenetic Classification of the Species of Anopheles (Culicidae: Anophelinae).

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.

Genetic diversity and transmission patterns of Echinococcus granulosussensu stricto among domestic ungulates of Sardinia, Italy

Cystic echinococcosis (CE), a parasitic zoonosis of public health and economic concern, is highly endemic in Sardinia, Italy. The study involved examining the intraspecific variability and demographic structure of Echinococcus granulosussensu stricto (s.s.) in common hosts of this parasite. Molecular surveillance included the fragment amplification of a partial mitochondrial gene, cox1 (750 bp), for a total of 69 isolates derived from sheep (n = 52), cattle (n = 11), pigs (n = 4), and goats (n = 2). It was ascertained that E. granulosus s.s. was the primary agent of infection among these ungulates and G1 genotype was highly prevalent (79.71%). Considerable intraspecific variation was found, revealing the existence of 22 haplotypes with relatively high haplotype (0.8555 ± 0.033) and low nucleotide diversities (0.00281 ± 0.00030). Population demographics indicated an expanding parasitic population signifying negative deviation from neutrality indices. Little genetic differentiation was found between the subpopulations of E. granulosus s.s. in the island. Moreover, the geographic dispersal of genotypes G1 and G3 also indicated similarity between Sardinian and mainland Echinococcus granulosus s.s. populations reaffirming the sympatric occurrence and efficient transmission of G1 and G3 genotypes. Molecular survey of CE has the potential to yield baseline information on the infective genotypes among the intermediate hosts and helps in devising suitable control strategies for curtailing the disease.

Low genetic variation in Echinococcus multilocularis from the Western Sichuan Plateau of China revealed by microsatellite and mitochondrial DNA markers

The prevalence of E. multilocularis is a major public health problem in China. To better understand the molecular epidemiology and evolutionary patterns of E. multilocularis, an adequate dataset regarding the genetic variance of this parasite is necessary. However, for now, available genetic data of E. multilocularis is still insufficient. In the study, the EmsB microsatellite and the partial mitochondrial cox1 gene were combined to investigate the genetic diversity of 64 E. multilocularis samples from human, dogs and voles. These samples were collected in the Western Sichuan Plateau, where the highest village-based human prevalence of alveolar echinococcosis was recorded worldwide. The aim of the study is to gather more informative genetic data of E. multilocularis in the areas, especially those obtained using the EmsB marker. The microsatellite analysis revealed 7 different EmsB profiles, 1 of which was found in 90.63% of the total samples collected from all 3 hosts. This major profile was identical to the one detected in the same area 16 years ago. The rest of the 6 profiles, each represented by only 1 isolate, did not correspond to any of the profiles previously reported. All the profiles detected in the study belonged to the Asian cluster. Meanwhile, according to sequence analysis of the 758 bp cox1 region, 4 haplotypes all assigned to the Asian clade were detected among the isolates. A star-like haplotype network was exhibited with a centrally positioned haplotype found in 93.75% of the samples. The overall haplotype and nucleotide diversities were both low. These findings provided evidence for a founder event or bottleneck and subsequent population expansion in E. multilocularis. The EmsB profiles were not fully consistent with the cox1 haplotypes. The same correspondence relationship was mainly observed in samples with the major profile P5 and the main haplotype EmHa1. A total of 54 isolates assigned to profile P5 were classified to the EmHa1 haplotype. In conclusion, both the microsatellite and mtDNA markers showed low variability within the Tibetan population of E. multilocularis. An EmsB profile and a cox1 haplotype were found to be predominant in the study area, which appears to remain steady for over a decade. The results reinforce the higher potential of the microsatellite DNA marker with high discriminative power to identify the very low genetic polymorphism of E. multilocularis than that of the partial cox1 sequencing. The data obtained in the study would be helpful to enlarge the data pool to further probe the possible origins and dispersal of E. multilocularis in China.

Morphological and molecular differentiation of Diplostomum spp. metacercariae from brain of minnows (Phoxinus phoxinus L.) in four populations of northern Europe and East Asia.

Metacercariae of trematodes from the genus Diplostomum are major helminth pathogens of freshwater fish, infecting the eye or the brain. The taxonomy of the genus Diplostomum is complicated, and has recently been based mainly on the molecular markers. In this study, we report the results of the morphological and molecular genetic analysis of diplostomid metacercaria from the brain of the minnow Phoxinus phoxinus from three populations in Fennoscandia (Northern Europe) and one population in Mongolia (East Asia). We obtained the data on the polymorphism of the partial mitochondrial cox1 gene and ribosomal ITS1-5.8S-ITS2 region of these parasites. РСА-based morphological analysis revealed that the parasites in the Asian and the European groups of Diplostomum sp. were distinctly different. Metacercariae from the brain of Mongolian minnows were much larger than those from the brain of Fennoscandian minnows but had much fewer excretory granules. Considering that the two study regions were separated by a distance of about 4500km, we also tested the genetic homogeneity of their host, the minnow, using the mitochondrial cytb gene. It was shown that Diplostomum-infected minnows from Mongolia and Fennoscandia represented two previously unknown separate phylogenetic lineages of the genus Phoxinus. Both molecular and morphological analysis demonstrated that the parasites from Fennoscandia belonged the species Diplostomum phoxini, while the parasites from Mongolia belonged to a separate species, Diplostomum sp. MТ.Each of the two studied Diplostomum spp. was associated with a specific, and previously unknown, genealogical lineage of its second intermediate host, P. phoxinus.

Hidden by the name: A new fluorescent pumpkin toadlet from the Brachycephalus ephippium group (Anura: Brachycephalidae).

Species of Brachycephalus has been having taxonomical issues due its morphological similarity and genetic conservatism. Herein, we describe a new species of Brachycephalus from the south Mantiqueira mountain range and semidecidual forests in the municipalities of Mogi das Cruzes, Campinas and Jundiaí, state of São Paulo, Brazil, based on an integrative approach. It can be distinguished from all species of the B. ephippium species group based on morphological characters (especially osteology and head shape), advertisement call and divergence in partial mitochondrial DNA gene sequences (16S). The new species is genetically similar to B. margaritatus and morphologically similar to B. ephippium. It can be differentiated from B. ephippium by the presence of dark faded spots on skull and post-cranial plates, presence of black connective tissue connective tissue scattered over dorsal musculature, parotic plate morphology, smaller snout-vent length (adult SVL: males 13.46–15.92 mm; females 16.04–17.69 mm) and 3% genetic distance. We also present natural history data and discuss the robustness of the integrative approach, geographic distribution, genetic data, behaviour, fluorescence in ontogeny, and conservation status.

Cystic echinococcosis in sheep and goats of Lebanon.

Cystic echinococcosis (CE), a zoonotic disease caused by the species complex of Echinococcus granulosus sensu lato (s.l.), is endemic in Middle East and the Mediterranean basin, where pastoral activity is widespread. Despite the chronic endemicity of the disease in Lebanon and neighbouring countries, recent data are scant. The objectives of this survey were to evaluate the current epidemiology of CE in Lebanon, investigate the prevalence and determine the infecting genotypes in locally raised sheep and goats. A multidimensional approach combining post-mortem inspection of slaughtered animals and molecular diagnosis of the parasite was conducted to this end. From 2018 to 2020, 62.9% of the sheep and 20.9% of the goats were found positive for CE. The presence of hydatids varied between organs, showing higher prevalence in the liver of sheep vs the lungs of goats, however, a higher fertility rate of hydatid cyst was observed in lungs for both animals. Molecular diagnosis based on partial mitochondrial cox1 gene (795 bp) showed that the majority of isolates were identified as E. granulosus sensu stricto (98.7%) and only one isolate from goat was identified as Echinococcus canadensis (genotype G7; 1.3%). Echinococcus granulosus s.s. population among the sheep and goats was represented by 22 haplotypes having very little genetic differentiation and relatively moderate haplotype diversity. Population demographics explored through neutrality indices suggested expanding population within the intermediate hosts. These results document the high prevalence of CE in the livestock of Lebanon and reveal for the first time the presence of three different genotypes G1, G3 and G7.

Phylogeography and shell morphology of the pelagic snail Limacina helicina in the Okhotsk Sea and western North Pacific

The pelagic snail Limacina helicina Phipps, 1774 is widely distributed in high-latitude seas and is a sensitive bioindicator of ocean acidification. It is known that the response patterns to ocean acidification differ among populations within or among species. Thus, it is important to understand their genetic population structure and identify the cryptic species. L. helicina displays different shell morphotypes, called “formae,” in different regions (North Atlantic, L. helicina forma helicina Phipps, 1774; Northwest Pacific, L. helicina f. acuta Spoel, 1967; Northeast Pacific, L. helicina f. pacifica Dall, 1871; Okhotsk Sea, L. helicina f. ochotensis Shkoldina, 1999: Spoel, 1967, pp. 257, 349). A recent study by Shimizu et al. (Journal of Molluscan Studies 84:30-37, 2018) showed clear genetic differentiation between two formae, L. helicina forma helicina (Svalbard in the Arctic Ocean) and L. helicina f. acuta (station K2 in the western North Pacific) (ΦCT = 0.59282), using partial mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. However, whether other formae of this species are genetically distinct remain unknown. Here we focused on L. helicina f. ochotensis, which inhabits the Sea of Okhotsk, and compared its shell morphology and partial mitochondrial COI gene sequences with another forma, L. helicina f. acuta, which is found in the western North Pacific. We found differences in the shell morphology of L. helicina f. ochotensis collected from the Sea of Okhotsk (off the coast of Monbetsu, Hokkaido, Japan) and L. helicina f. acuta collected from the western North Pacific (station K2). However, molecular analysis of an mtDNA COI region (503 bp) revealed that most individuals collected from Monbetsu (Sea of Okhotsk) were haplotype 1 (75.5%), which is also the major haplotype found in L. helicina f. acuta (western North Pacific). Our results suggest that individuals of L. helicina are frequently dispersed between the Sea of Okhotsk and the western North Pacific by ocean currents. Thus, the two formae, L. helicina f. acuta and L. helicina f. ochotensis, are more likely to be attributable to phenotypic plasticity in response to different environmental parameters, such as temperature and salinity, than to differences in their genetic backgrounds.

Mitohelper: A mitochondrial reference sequence analysis tool for fish eDNA studies

AbstractFish eDNA metabarcoding is a nonintrusive, time‐ and cost‐effective way to detect biodiversity, with potential applications in ecosystem‐based management and fisheries assessment. Nevertheless, fish‐specific eDNA resources are currently not well‐developed, and many fish species are not yet sequenced in reference databases. We developed Mitohelper, a Python‐based command line tool to annotate and align fish mitochondrial sequences available in the existing MitoFish database. Mitohelper improves MitoFish annotations by adding gene names and additional taxonomic classifications. Using these improved reference datasets, Mitohelper's getrecord function searches MitoFish for available mitochondrial (cytochrome c oxidase, 12S rRNA, and others) gene sequences against a user‐provided list of fish taxonomic names. Mitohelper's getalignment function aligns (often partial) mitochondrial gene sequences to a user‐specified full‐length reference sequence for the assessment and visualization of overlapping sequencing regions. To facilitate the development of taxonomic classifiers, we combined Mitohelper's 12S rRNA dataset with SILVA’s 16S rRNA and 18S rRNA datasets in a QIIME 2‐compatible format. By providing valuable information on taxonomic and gene region coverage of currently available fish mitochondrial data, Mitohelper's functions promote informed experimental design that can guide sequencing and analysis strategies. In summary, Mitohelper improves the breadth and functionality of eDNA data resources, as well as the accuracy of taxonomic classification. Mitohelper and its reference datasets are updated approximately monthly and available at https://github.com/aomlomics/mitohelper.

Locomotor responses to salt stress in native and invasive mud-tidal gastropod populations (Batillaria).

Plasticity in salt tolerance can be crucial for successful biological invasions of novel habitats by marine gastropods. The intertidal snail Batillaria attramentaria, which is native to East Asia but invaded the western shores of North America from Japan 80 years ago, provides an opportunity to examine how environmental salinity may shape behavioral and morphological traits. In this study, we compared the movement distance of four B. attramentaria populations from native (Korea and Japan) and introduced (United States) habitats under various salinity levels (13, 23, 33, and 43 PSU) during 30 days of exposure in the lab. We sequenced a partial mitochondrial CO1 gene to infer phylogenetic relationships among populations and confirmed two divergent mitochondrial lineages constituting our sample sets. Using a statistical model‐selection approach, we investigated the effects of geographic distribution and genetic composition on locomotor performance in response to salt stress. Snails exposed to acute low salinity (13 PSU) reduced their locomotion and were unable to perform at their normal level (the moving pace of snails exposed to 33 PSU). We did not detect any meaningful differences in locomotor response to salt stress between the two genetic lineages or between the native snails (Japan vs. Korea populations), but we found significant locomotor differences between the native and introduced groups (Japan or Korea vs. the United States). We suggest that the greater magnitude of tidal salinity fluctuation at the US location may have influenced locomotor responses to salt stress in introduced snails.

First Report of Heterodera trifolii on White Clover and Rumex obtusifolius in Costa Rica

In 2018, during a survey sampling for cyst-forming nematodes, two populations of Heterodera sp. were found in the north region of Cartago (Llano Grande and Oreamuno), Costa Rica. White females and cysts were attached to the plant roots of two weeds, white clover (Trifolium repens L.) and Rumex obtusifolius L. Plants were asymptomatic aboveground. Cysts were extracted from soil samples using the Fenwick method (Fenwick 1940) and cut to release the second-stage juveniles (J2s). Cysts were lemon-shaped with a prominent vulval cone, light to dark brown coloring, and ambifenestrate with developed underbridge and bifurcated at the end. Measurements combining both populations are given in micrometers and provided in the following format: mean ± SD (range). Cysts (n = 20): length excluding neck = 744.3 ± 61.6 (648.5 to 837.4); width = 495.9 ± 60.5 (421.9 to 621.1); fenestral length = 50.7 ± 6.4 (40.3 to 61.3); and semifenestral width = 36.2 ± 5.6 (24.1 to 45.7). J2s (n = 60): length = 541.1 ± 25.6 (492.5 to 604.9); width = 22.9 ± 1.7 (20.5 to 27.3); stylet = 27.9 ± 0.9 (26.2 to 29.6); labial region height = 4.7 ± 0.3 (3.8 to 5.5); labial region diameter = 9.4 ± 0.5 (8.5 to 10.9); DGO = 5.9 ± 0.5 (4.6 to 7.3); anterior end to excretory pore = 122.2 ± 8.3 (106.4 to 148.7) and to median bulb valve = 81.2 ± 5.3 (67.3 to 92.6); tail length = 66.6 ± 4.9 (55.9 to 77.3); and hyaline region of tail length = 37.1 ± 3.7 (27.7 to 44.5). No males were found. The morphological and morphometrical features of the two Heterodera populations overlapped with each other and within other H. trifolii populations reported worldwide (Sekimoto et al. 2017; Subbotin et al. 2010). The primer sets TW81/AB28 (Subbotin et al. 2001), D2A/D3B (De Ley et al. 1999), and HTcox1F2 (5′-GCTTCTGATCTTTCCTTTCCACGTA-3′, this study)/JB4 (Derycke et al. 2005) were used for amplification of the internal transcribed spacer (ITS) rRNA, the D2-D3 expansion segments of the 28S rRNA (28S), and the partial mitochondrial cox1 gene (cox1), respectively. The resulting sequences were submitted to GenBank (ITS, MT020783; 28S, MT010295; and cox1, MT007852). A BLASTn search of the ITS, 28S, and cox1 gene sequences of H. trifolii from Costa Rica revealed a 100% identity with sequences of H. trifolii from Japan (LC208684), South Korea (MN720070), and the United States (MK093174), respectively. A greenhouse essay was established to confirm the reproduction of H. trifolii on each host. Using 10 replicates (individual pots) per treatment (hosts), pots were inoculated with 1,000 individuals (eggs + infective J2s). The experiment was conducted twice. The average greenhouse temperature was 22.1 ± 3.8°C, and plants were harvested 75 days after inoculation. Results showed that H. trifolii was able to reproduce successfully on both hosts. Final average population (roots + soil) in R. obtusifolius and T. repens was 21,516 and 2,626 nematodes (all stages and cysts), respectively. These two weed species can be a source of inoculum for economically important crops. Therefore, studies on life cycle and pathogenicity assays are being performed on agricultural crops. To our knowledge, this is the first report of H. trifolii on white clover and R. obtusifolius in Costa Rica.