Cytochrome C Oxidase Subunit I Research Articles

COI DNA barcoding to differentiate Haemagogus janthinomys and Haemagogus capricornii (Diptera: Culicidae) mosquitoes

The genus Haemagogus (Diptera: Culicidae) includes species that are important vectors of pathogens such as the yellow fever virus. The accurate identification of these species is essential for the control of zoonoses. Females of Hg. capricornii and Hg. janthinomys are morphologically indistinguishable, which makes the use of alternative identification techniques desirable. This study aimed to obtain sequences of the mitochondrial cytochrome c oxidase I (COI) gene, in the region widely used for DNA barcoding, of Haemagogus specimens from the state of São Paulo, Brazil, to evaluate the effectiveness of these sequences in the molecular identification of the species. A total of 37 female and 2 male mosquitoes were collected in various locations in the state of São Paulo, using methods such as hand-nets, Shannon traps, CDC light traps with CO2 bait and Nasci aspirators. The sequences of a 710 bp fragment of the COI gene were amplified by PCR and sequenced. A phylogenetic tree reconstruction was conducted using the Bayesian approach implemented in MrBayes v3.2.2, providing support values for taxa where genetic clusters may indicate the presence of new or cryptic species. We obtained 39 COI sequences representing three species: Haemagogus capricornii, Haemagogus leucocelaenus, and Haemagogus janthinomys. Bayesian analysis of the sequences produced clades that corroborate the morphological identification of the species. The separation of Hg. capricornii and Hg. janthinomys received 100 % statistical support and the Hg. capricornii was very well supported (91 %). The two sequences from male specimens, morphologically identified as Hg. capricornii, were grouped in the same clade, a sister clade of Hg. janthinomys. It is important to highlight that the Hg. janthinomys were positioned in several subclades, showing a polymorphism of this species within the state, a situation not observed for Hg. capricornii. For the first time, sequences of the mtCOI gene from Hg. capricornii were obtained and related to morphologically identified specimens. COI sequences proved effective in the molecular identification of Haemagogus species. This study contributes to the expansion of the GenBank database, providing the first sequences of Hg. capricornii and new sequences for Hg. janthinomys and Hg. leucocelaenus.

DNA barcoding and new record of Arcynopteryx dichroa (McLachlan, 1872) (Plecoptera: Perlodidae) from Serbia

Arcynopteryx dichroa (McLachlan, 1872) (Perlodidae) is recorded for the first time from southwestern Serbia. It was collected in the Djerekar Spring (the Pešter Plateau). Previously, this species was found in Timok River in the eastern part of the country, with the last known record dating back to 1995. Identification of the collected specimens was confirmed using the nucleotide sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. A diagnosis of the larval characters is provided, and species distribution is discussed.

A Microcosm Analysis of Species-Specific Responses of Chironomidae on Heavy Metal Pollution in The Nyanza Gulf of Lake Victoria

The Chironomidae family, known as "non-biting midges" in their adult stage and "bloodworms" in their larval stage, consists of diverse dipteran insects inhabiting various global aquatic environments. Despite extensive global research, data on Chironomidae in the polluted Nyanza Gulf of Lake Victoria, Kenya, is scarce, and molecular identification methods have not been explored. This study aimed to quantify heavy metal concentrations in water, sediment, and insect samples and assess their impact on Chironomid species identified using mitochondrial DNA barcoding of the cytochrome oxidase subunit 1 (COI) gene. Analysis of Variance was used to determine if there were any statistically significant differences in heavy metal concentrations across different sample types or locations along the pollution gradient. Chironomids were collected from Nyanza Gulf, focusing on a pollution gradient. Results showed that concentrations of arsenic (As), lead (Pb), and cadmium (Cd) in insect, water, and sediment samples exceeded standard limits, while mercury (Hg) concentrations were within limits. Significant variations (p ≤ 0.05) in Pb levels were observed in water samples, and heavy metal concentrations in sediment samples varied significantly (p ≤ 0.05), with Pb showing the highest variation (p ≤ 0.0001). Insect samples exhibited significant differences (p ≤ 0.0001) in As and Hg contents. Genetic analysis identified two known species: Chironomus transvaalensis at the heavily polluted Kisumu station and Chironomus pseudothummi at the moderately polluted Kendu Bay and Homa Bay stations. Additionally, a unique Chironomus species was found on Ndere Island, a relatively clean site with restricted human activities. Sequence comparisons indicated proximity to global data but also highlighted the evolutionary significance and uniqueness of the identified species. This study demonstrated the potential use of genetic methods in determining Chironomid species diversity, community structure, and abundance in relation to heavy metal concentration. It suggests that heavy metal pollution may act as a selective pressure, driving the evolution of Chironomid species. The study recommends combining genetic approaches with other pollution sources for a comprehensive understanding of using this species in monitoring pollution

Molecular identification of Nemacheilus sp. from Bangka Island, Indonesia Based on the Cytochrome C oxidase Subunit I (COI) Gene

Abstract Nemacheilus is a genus of stone loaches native to Asia. There are 13 species of the genus Nemacheilus that can be found in western Indonesia, from 44 species that exist in Asia. During our expedition to Bangka Island, which took place between the 20th and the 25th of January 2023, we discovered 15 live specimens of Nemacheilus sp. These specimens were collected using a fish trap in the Bencah River, Bangka Island, Indonesia. We identified Nemacheilus sp based molecular analysis by using the DNA Barcoding method based on the Cytochrome C oxidase Subunit I (COI) gene. DNA barcoding is a solution to provide accurate, fast, and automatable identification of species and species discovery. Based aon the COI gene, Nemacheilus sp. Nemacheilus from Bangka Island is genetically closest to Nemacheilus pfeifferae, which is found in Sumatra. Sumatra and Bangka Island have a history of river connection before the maximum glacial phase II. However, based on the similarity of DNA base pairs, these two species are only 90% similar. This indicates that the genetic difference between these two species is 10%, which is far below the threshold for what is considered to be an identical species, which is under 3%. Therefore, we hypothesize that the Nemacheilus that was found on the island of Bangka is not the species known as Nemacheilus pfeifferae, but rather another species. Since the data for this other species are not yet available in Genbank, we are referring to it as Nemacheilus sp. Bangka for the time being.

Molecular phylogeny and species diversity of the genus Dichaetophora Duda and related taxa (Diptera: Drosophilidae)

Our intensive surveys of wild drosophilids in East and Southeast Asia discovered a great species diversity (more than 100 putatively new species) of the genus Dichaetophora, which is currently comprised of 67 formally described species assigned into five species groups, i.e., agbo, tenuicauda, acutissima, sinensis and trilobita. In the present study, we delimited species from a huge amount of samples of Dichaetophora and allied taxa (the genus Mulgravea and the subgenus Dudaica of Drosophila) collected from a wide range of the Oriental and east Palearctic regions. We first sorted all specimens into morpho-species, and representative specimen(s) selected from each morpho-species were subjected to barcoding of COI (the cytochrome c oxidase subunit I gene) sequences. The applied ASAP (Assemble Species by Automatic Partitioning) analysis estimated a total of 166 to 168 MOTUs (molecular operational taxonomic units). Integrating this result with morphological evidence from re-examined, detailed structures of male terminalia, we recognized a total of 144 (109 new and 35 known) species in our sample. Out of them, 83 species representing the supraspecific taxa of Dichaetophora, Mulgravea and Dudaica were selected, along with 33 species from major genera and subgenera of Drosophila in the tribe Drosophilini, as in-group and four species from the tribe Colocasiomyini as out-group for phylogenetic reconstruction based on 12 nuclear gene markers. In the trees constructed by the maximum likelihood and Bayesian inference methods, the three focal taxa (i.e., Dichaetophora, Mulgravea and Dudaica) formed a clade provisionally called the “pan-Dichaetophora”. Within this large clade, the agbo, tenuicauda, sinensis and trilobita groups of Dichaetophora, Mulgravea and Dudaica were recovered as monophyletic groups, but Dichaetophora and its acutissima group were regarded as paraphyletic. In addition, two clusters were recognized among ungrouped species of Dichaetophora. Thus, the present study has uncovered some issues concerning the taxonomy of the pan-Dichaetophora. Such issues will be addressed elsewhere in the phylogenetic reclassification of the pan-Dichaetophora, along with descriptions/redescriptions of a large number of new/known species delimited in the present study.

DNA Barcoding of the Endemic Freshwater Catfish Encheloclarias tapeinopterus (Bleeker, 1853) (Siluriformes:Clariidae) from Bangka Island, Indonesia

Abstract Encheloclarias tapeinopterus, an endemic catfish exclusively found in Bangka Island, Indonesia. The population of this species has been observed to be decreasing at an alarming rate, which has resulted in the IUCN Red List classifying it as a species that is vulnerable (VU). During our expedition on the 25th and 26th of January 2023, a total of Two specimens of E. tapeinopterus were collected from Kelumpik River, Pinang Sebatang Village, Central Bangka Regency, Bangka Island, Indonesia. In this groundbreaking study, we proudly present the pioneering documentation of E. tapeinopterus DNA barcoding, focusing on the Cytochrome C oxidase Subunit I (COI) gene. The recorded DNA Barcode of E. tapeinopterus has been officially registered in the Genbank under the accession code OQ281707. By employing DNA barcoding, we offer an advanced and expedient method for precise species identification and discovery, revolutionizing the field of taxonomy. Phylogenetic analysis revealed that E. tapeinopterus forms a distinct clade alongside the Clarias and Heterobranchus genus, shedding light on its evolutionary relationships and genetic affinities.

Generalist species exhibit more genetic structure in comparison to a habitat specialist: Evidence from a phylogeographic study of two freshwater crabs (Decapoda: Potamonautidae: Potamonautes), implications for habitat conservation.

During the present study, the phylogeography of the wetland specialist, freshwater crab, Potamonautes flavusjo, was investigated and compared to that of the ubiquitous, generalist, P. sidneyi, using DNA sequence data from the mitochondrial cytochrome oxidase subunit one (COI). We inferred the evolutionary history of each species and compared their population-level genetic structure by constructing haplotype networks and using an analysis of molecular variation. Additionally, we explored the evolutionary relationship between southern African lentic and lotic freshwater crab species by examining the usefulness of carapace attributes in relation to genetic indices and a species' assumed dispersal capacity. In the lentic species, P. flavusjo, a single interconnected haplocluster characterized by shared haplotypes was observed, suggesting marked maternal dispersal, a result corroborated by the low F ST values. In contrast, for the lotic species, P. sidneyi two distinct haploclusters and marked genetic differentiation was observed indicating the absence of maternal dispersal, a result corroborated by the high F ST values. The phylogenetic relationship in P. sidneyi was further investigated using a maximum likelihood and Bayesian inference analyses with the addition of sequence data from the mitochondrial 16S rRNA locus to estimate divergence times. Three species delimitation methods (ASAP, PTP, and bGMYC) were used to explore the presence of distinct lineages in P. sidneyi. The phylogenetic results indicated that within P. sidneyi two clades were present, while divergence time estimates suggest cladogenesis during the middle to late Pleistocene. The species delimitation methods used showed moderate congruence, however all oversplit the number of putative lineages. Our results indicated that P. sidneyi is a species complex comprised of two cryptic lineages, occurring in sympatry contemporarily possibly alluding to secondary contact. Carapace height was found to have no discernible influence on the genetic indices and presumed dispersal capabilities of mainland southern African freshwater crab species. The importance of our results are discussed in terms of conservation of freshwater habitats.

Assessing the Potential of Tortistilus (Hemiptera: Membracidae) from Northern California Vineyards as Vector Candidates of Grapevine Red Blotch Virus.

Ceresini treehoppers are present in northern California vineyard ecosystems, including the closely related Spissistilus and Tortistilus (Hemiptera: Membracidae). These membracids are not direct pests of wine grapes, but S. festinus is a vector of grapevine red blotch virus (GRBV). No information is available on the ability of Tortistilus spp. to transmit GRBV. In this study, Tortistilus were collected on yellow panel cards across 102 vineyard sites and surrounding areas in Napa Valley, California, USA in 2021-2023. Specimens were morphotyped, sexed and tested for GRBV ingestion and acquisition by multiplex PCR or qPCR. Phylogenetic analysis of the partial sequence of mt-COI and ITS gene fragments of a subset of 40 Tortistilus specimens revealed clustering in a monophyletic clade with T. wickhami with the former barcode sequence. Only 6% (48/758) of the T. wickhami tested positive for GRBV, but none of the heads with salivary glands (0%, 0/50) of the dissected specimens tested positive for GRBV, indicating no virus acquisition. In contrast, half of the dissected heads with salivary glands of S. festinus (52%, 12/23), from the same collection vineyard sites, tested positive for GRBV. Together, our findings confirmed the presence of T. wickhami in northern California vineyards and suggested a dubious role of this treehopper as a vector of GRBV.

Genetic Diversity and Population Structure of Amphidromous Goby (Stiphodon semoni) in Western Part of Southern Java Waters

Graphical Abstract Research Highlight The genetic diversity of Stiphodon semoni is high. All populations have high haplotype diversity except for Cimaja. No genetic structure is observed within the populations of semoni. The population of semoni has undergone demographic expansion. Cibareno River has the potential to be a protected area. Abstract Fishing activities negatively impact fish populations, potentially causing a decline in fish stocks. Nevertheless, ensuring diversity and connectivity among populations can mitigate these adverse effects. To evaluate the connectivity of river mouths in the western part of Southern Java waters, we sequenced forty Stiphodon semoni individuals from five populations using mitochondrial cytochrome oxidase subunit 1 as molecular markers. The study revealed that S. semoni populations showed high diversity (0.821), with the population in Cimaja displaying the lowest diversity (0.464). Furthermore, the result of the analysis of molecular variance was a Fst value of 0.0630 with a p-value of 0.22. These results along with the result of the haplotype network indicated no significant genetic differences among these populations. This implies that the river mouths in the western part of Southern Java waters are interconnected. The distribution of mismatches showed a single peak, indicating that the populations have undergone demographic expansion. This information could be valuable for the conservation and management of S. semoni in the western part of the Southern Java waters.

Evaluation of the Effects of Papain on Schistosoma mansoni: Miracidial Infection Capacity, Infection Prevalence, Cercarial Shedding and Molecular Changes in Biomphalaria alexandrina.

The aim of the present study is to assess the molluscicidal, larvicidal and genotoxicological activities of papain and how it can affect the host-parasite interactions. Toxicity of papain on snails by making series of concentrations to calculate LC50, and then study its larvicide effect on the free larval stages of S. mansoni and infection rate of snails. Papain has a molluscicidal activity on adult snails of Biomphalaria alexandrina with a lethal concentration LC50 equals to 43.1mg/L. In addition, it has activity on miracidia with half Lethal time (LT50) of 16.11min., and on cercariae with 12.1min. compared to control ones. The sub lethal concentration LC10 and LC25 (6.9 or 24.1mg/L, respectively) decreased the survival rate of snails at the first cercarial shedding, the rate of infection, the average total number of cercariae per snail, the shedding period and the life span of snails, while the prepatent period was significantly increased than the control ones. The morphological alterations in cercariae after exposure to papain were occurred where the cercariae lacked motility and some had a dark tail with complete detachment of head and tail. Compared to the control group, the levels of cytochrome oxidase subunit I (COI) and (ND1) genes significantly decreased in snails after exposure to papain. Papain could be used as a potential molluscicide for elimination of schistosomiasis and decrease its transmission and deterioration of host-parasite interaction.

Multi-marker DNA metabarcoding for precise species identification in ichthyoplankton samples

Ichthyoplankton monitoring is crucial for stock assessments, offering insights into spawning grounds, stock size, seasons, recruitment, and changes in regional ichthyofauna. This study evaluates the efficiency of multi-marker DNA metabarcoding using mitochondrial cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA gene markers, in comparison to morphology-based methods for fish species identification in ichthyoplankton samples. Two transects with four coastal distance categories were sampled along the southern coast of Portugal, being each sample divided for molecular and morphological analyses. A total of 76 fish species were identified by both approaches, with DNA metabarcoding overperforming morphology—75 versus 11 species-level identifications. Linking species-level DNA identifications with higher taxonomic morphological identifications resolved several uncertainties associated with traditional methods. Multi-marker DNA metabarcoding improved fish species detection by 20–36% compared to using a single marker/amplicon, and identified 38 species in common, reinforcing the validity of our results. PERMANOVA analysis revealed significant differences in species communities based on the primer set employed, transect location, and distance from the coast. Our findings underscore the potential of DNA metabarcoding to assess ichthyoplankton diversity and suggest that its integration into routine surveys could enhance the accuracy and comprehensiveness of fish stock assessments.

Unveiled species diversity of moss-feeding mites (Stigmaeidae: Eustigmaeus): a research on their distribution, habitat, and host plant use in Japan.

The genus Eustigmaeus Berlese, 1910 represents the unique phytophagous group within the superfamily Raphignathoidea. Four species within this genus have been known to inhabit mosses and feed on them as larvae, nymphs, and adults. However, the interactions with mosses have remained poorly understood. In order to reveal the diversity and host-plant use of the moss-feeding species, we conducted an extensive field study in Japan. This study revealed an array of moss-feeding species inhabiting various moss species, with 10 morphologically distinctive species newly documented in Japan. Through DNA barcoding based on cytochrome c oxidase subunit I (COI) sequences, these morphospecies were recovered as distinct entities. Notably, the host-plant use of four species was elucidated. Among these, Eustigmaeus sp. 9 exhibited polyphagy, while three species (Eustigmaeus spp. 1-3) demonstrated varying degrees of host specificity, each using moss species from the Hypnales, Philonotis, and Dicranidae, respectively. While a few moss-feeding species were frequently found in the same geographic area, more than one species rarely co-occurred within the same moss colonies. Eustigmaeus offers a unique study system, with its diverse moss-feeding species and indications of specific host plant use. Consequently, the moss-feeding Eustigmaeus serves as a valuable model for exploring the macroevolutionary patterns underlying diversification in moss-feeding arthropods.

Identify Lumbricus Rubellus According DNA Barcoding

The species of earthworms is usually determined through careful observation of morphological features, is often accomplished by closely examining morphological characteristics, which are frequently sexual characteristics only seen in mature individuals. Earthworm species identification as such, it is sometimes hard to identify juveniles or cocoons, which might introduce bias into studies that record species richness and abundance. A viable method for species discrimination is DNA barcoding, which uses a brief, standardized DNA fragment for species identification, use of DNA remains the best in determining the species. Lumbricus rubellus have been found on in the common epigeic earthworm from Iraq in Al-Diwaniyah city, the presence of this worm is recorded for the first time in Iraq, specifically in the city of Diwaniyah. This study tested sequence data for the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene in order to identify the utility of DNA barcodes in the identification of earthworm species.

Copula lucentia sp. nov., a new box jellyfish (Cnidaria: Cubozoa: Carybdeida) from Western Mediterranean Sea

A new species of box jellyfish, Copula lucentia sp. nov., is described from El Campello, Spain, and compared to the holotype of Copula sivickisi that was successfully recovered after being lost for almost 60 years. So far, the only cubozoan species recorded in the Western Mediterranean was Carybdea marsupialis. The genus Copula just included the type species (Copula sivickisi) reported from different localities in the Pacific, Atlantic, and Indian Oceans. Morphologically, this new species possesses the typical characteristics of the genus Copula (four adhesive pads on the apex and a vertical keyhole-shaped rhopalial niche ostium) but it can be differentiated from the C. sivickisi holotype by the velarial canal pattern. In C. lucentia sp. nov., the velarial canal roots taper towards the velarial rim and each root bears one to two narrow triangular canals with sharp tips, resembling a bird beak. In contrast, the velarial canal roots of C. sivickisi bear two short, broad canals that increase breadth towards the velarial rim and split up into three- to six-lobed, finger-like canals with rounded tips, giving the canal pattern a paw-like appearance. Molecular analyses of mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 16S ribosomal DNA genes confirm the distinction of the new species and show that there are even more Copula species than expected, but which will not be focused on here. This study highlights not only that there are species yet to be discovered in the Mediterranean Sea but that a revision of the genus Copula is necessary.

Taxonomy and DNA barcoding of the dark-winged fungus gnat genus Zygoneura Meigen (Diptera: Sciaridae) from China, with revision of the type materials

The genus Zygoneura Meigen is revised thoroughly from China, and 14 species are recognized and illustrated, including four new species: Zygoneura (Allozygoneura) xizangensis Shah & Huang sp. nov., Zygoneura (Pharetratula) minuscula sp. nov., Zygoneura (Pharetratula) motuoensis sp. nov. and Zygoneura (Pharetratula) yangi sp. nov. In addition, Zygoneura (Pharetratula) divergens (Mamaev), Zygoneura (Pharetratula) flavicornis (Mamaev), and Zygoneura (Pharetratula) subdivergens (Mohrig & Mamaev) are reported for the first time from China. The identification of these species is supported by both morphological characteristics and sequence data obtained from cytochrome oxidase subunit one (COI) in the DNA barcode analysis. Furthermore, a checklist of the known Zygoneura species in China is also provided, along with an identification key for males.

Candidatus Midichloria mitochondrii can be vertically transmitted in Hyalomma anatolicum

In this study, a tick intracellular symbiont, Candidatus Midichloria mitochondrii, was detected in Hyalomma anatolicum from Xinjiang, China. Morphological identification and cytochrome oxidase subunit I sequence alignment were used for molecular identification of the tick species. PCR detection further revealed the presence of endosymbiont C. M. mitochondrii in the tick. Specific primers were designed for Groel and 16S rRNA genes of C. M. mitochondrii for PCR amplification and phylogenetic analysis. To further investigate the vertical transmission characteristics of C. M. mitochondrii, specific primers were designed based on the FabⅠ gene fragment to detect C. M. mitochondrii in different developmental stages and organs of the tick using qPCR. Of the 336 tick specimens collected from the field, 266 samples were identified as H. anatolicum on the basis of morphological characteristics. The gene fragment alignment results of COI confirmed that these ticks were H. anatolicum. The phylogenetic analysis showed that Groel gene of C. M. mitochondrii clustered with Midichloria strains detected in Ixodes ricinus ticks from Italy and Ixodes holocyclus ticks from Australia, with 100% sequence similarity. Furthermore, the 16S rRNA gene of C. M. mitochondrii clusters with the strains isolated from Hyalomma rufipes ticks in Italy, exhibiting the highest degree of homology. qPCR results showed that C. M. mitochondrii was present at all developmental stages of H. anatolicum, with the highest relative abundance in eggs, and lower relative abundance in nymphs and unfed males. With female tick blood feeding, the relative abundance of C. M. mitochondrii increased, and a particularly high relative abundance was detected in the ovaries of engorged female ticks. This study provides information for studying the survival adaptability of H. anatolicum, and provides data for further investigation of the mechanisms regulating tick endosymbionts in ticks, enriching the reference materials for comprehensive prevention and control of tick-borne diseases.

DNA Barcoding of Freshwater Eels Anguilla spp from Kuari River Based on Cytochrome C Oxidase Subunit I (COI) Gene

Graphical Abstract Highlight Research Freshwater eels in Kuari River consisted of two species marmorata and A. bengalensis Anguilla marmorata had the highest similarity of 99.82%-100%, while bengalensis indicated the highest identity of 99.84%-100% to the same species in the GenBank database. Phylogenetic species indicated that Anguilla marmorata and Anguilla bengalensis form two different sub-clusters. The water quality in the Bengkulu Kuari River were still in tolerance for fish growth and survival. Abstract Freshwater eels (Anguilla spp.) are classified in the family Anguillidae, and included in the catadromous group. This study aimed to determine the COI gene sequences of the mitochondrial DNA, analyze the genetic distances, and phylogenetics, and characterize the physical and chemical parameters of between freshwater eels habitat in the Kuari River Bengkulu. This research was conducted from November 2020 – April 2021. The methods used in barcoding eel species were DNA isolation, DNA amplification using PCR (Polymerase Chain Reaction), electrophoresis, and sequencing of COI gene regions in mtDNA. The COI mtDNA gene fragments were obtained from PCR results with an annealing temperature of 50°C for 30 seconds in 35 cycles. BLASTn analysis of eel samples AM3 and AM4 had the highest similarity of 99.82%-100% to Anguilla marmorata, and samples AB2-AB5 indicated the highest identity of 99.84%-100% to Anguilla bengalensis. Phylogenetic species indicated that Anguilla marmorata and Anguilla bengalensis form two different sub-clusters. The water qualities in the Bengkulu Kuari River were temperatures 26.5-27.5oC, pH 7.1-8.7, dissolved oxygen 6.19-9.54 mg L-1, brightness 21-47 cm, ammonia 0.16-0.41 mg L-1, total alkalinity 20–52 mg L-1, TDS 33–49 mg L-1, salinity 0.3-0.4 ppt and water velocity 0.5-0.8 ms-1. The COI gene in DNA barcoding is very appropriate to be used for the identification of Anguilla spp species by comparing the DNA sequence of the COI gene with the existing database in the Genbank.

Detection Limits of Insect Fragments in Spiked Whole Wheat Flour Using Multiplex Polymerase Chain Reaction (PCR)

The need for a sensitive molecular method to detect specific species of insect contaminants in food products remains a significant challenge in the food industry. This study evaluated the detection limit of a multiplex end-point PCR assay for detecting insects in food. The assay amplifies two fragments of the cytochrome oxidase subunit I gene (COI-Fa and COI-Fb) and one fragment of the protein-coding wingless (wg) gene found in insects. Five insect species, comprising three vectors of foodborne pathogens (the housefly, Musca domestica, the American cockroach, Periplaneta americana, and the pharaoh ant, Monomorium pharaonis) and two storage insect pests (the red flour beetle, Tribolium castaneum and the Indian meal moth, Plodia interpunctella), were spiked separately and in combination at levels of 1, 0.1, 0.01, and 0.001% in whole wheat flour. At spike levels greater than 0.01%, amplicon bands of expected sizes were seen in 100% of samples containing fragments from distinct insect species. At least 25% of spiked samples at the lowest spike level had amplicon bands, except for samples spiked with M. domestica. Results showed an 18.9% probability (with 11.3% and 30% lower and upper confidence limits, respectively) of detecting insect fragments at the lowest spike level (0.001%, corresponding to 3–22 fragments), which is far below the FDA’s regulatory level of less than 75 fragments per 50 g of wheat flour. The intensity of amplicon bands in the gel images was higher at higher spike levels. However, this method is not quantitative enough to extrapolate the intensity of the amplicon bands to the number of insect fragments present in a sample. This multiplex assay was also evaluated in a variety of market food samples derived from plants and animals, showing its potential use in various food types. Overall, the sensitivity and specificity of this molecular approach suggest that it could be used in the future as a screening tool for detecting insect contaminants in food.

Arthropod diversity in shallow subterranean habitats of the Appalachian Mountains

Subterranean arthropods are important components of soils and contribute essential food-web functions and other ecosystem services, however, their diversity and community composition has scarcely been assessed. Subterranean pitfall traps are a commonly used method for sampling soil habitats in Europe but have never been widely implemented in the Americas. We used subterranean pitfall traps to sample previously unsurveyed arthropod communities in southwestern Virginia, U.S. Traps were placed in shallow subterranean habitats (SSHs), underground habitats close to the surface where light does not penetrate, and more specifically at the interface between the soil and underlying “milieu souterrain superficiel”—a microhabitat consisting of the air-filled interstitial spaces between rocks (abbreviated MSS). In total, 2,260 arthropod specimens were collected constituting 345 morphospecies from 8 classes, 33 orders, and 94 families. A region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified and sequenced, and objective sequence clustering of 3% was used to establish molecular operational taxonomic units (mOTUs) to infer observed species richness. In all, 272 COI barcodes representing 256 mOTUs were documented for rare soil-dwelling arthropod taxa and are published to build a molecular library for future research in this system. This work is the first taxonomically extensive survey of North American soil-dwelling arthropods greater than 10 cm below the soil surface.

Nematodes of concern to public and animal health associated with the invasive snail Achatina fulica, in Sergipe, Northeastern Brazil

The giant African land snail Achatina fulica is known to be the intermediate host of a number of nematode species that are prejudicial to human and veterinary health, being also an agricultural and urban pest. The present study investigated the presence of nematodes in A. fulica and other terrestrial mollusks in 24 municipalities of Sergipe State, northeastern of Brazil, in the dry and rainy seasons. In 2019 and 2020, the specimens were collected in standard 20 m × 10 m plots (10 min/01 collector), while in 2021, they were collected by convenience sampling. The nematode species were identified based on sequencing of the mitochondrial Cytochrome C Oxidase Subunit I (COI) gene and the ribosomal nuclear Internal Transcribed Spacer (ITS 2). Specimens of A. fulica infected with Angiostrongylus cantonensis, a nematode that causes eosinophilic meningitis (EM) in humans and animals, were collected in four municipalities. Two nematodes of veterinary importance were also identified, Aelurostrongylus abstrusus and Cruzia tentaculata in seven municipalities. This is the first record of Cruzia tentaculata in Sergipe. Free-living nematodes were also found in 18 municipalities. One of these species – Caenorhabditis briggsae – was identified in the municipality of Itabaiana. Specimens of other mollusks species were also collected. Only one of these mollusks, C. fasciata from Japaratuba, was found infected with free-living nematodes. A. fulica showed greater positivity for nematodes in the dry season, especially in the regions of Lower São Francisco River, Greater Aracaju and South Central Sergipe. The probability of infection increased when mollusks were larger and more recurrent in plots. Given that A. cantonensis is the etiological agent of EM and was found associated to A. fulica in four municipalities, it will be important to implement programs of continuous monitoring of the mollusk's fauna, combined with educational programs that provide information necessary to control A. fulica.