Mitochondrial 16S rRNA Gene Research Articles

Description of a new species of the Asian newt genus Tylototriton (Amphibia, Urodela, Salamandridae) from Hunan Province, China

The newt genus Tylototriton is widely distributed in east, southeast, and south Asia. Previous studies have shown that basic surveys of this genus are far from comprehensive, and unknown species still exist within the group. In this study, we described a new species of this genus, Tylototriton gaowangjienensissp. nov., from Gaowangjie National Nature Reserve in Hunan Province, China, based on evidence from molecular identification and morphological comparisons. Additionally, we conducted phylogenetic analysis of the genus to elucidate the taxonomic status of the new species. Based on combined mitochondrial 16S rRNA and ND2 gene sequences, the phylogenetic tree supported the new species as a member of the T. wenxianensis species subclade in the subgenus Yaotriton. It can be distinguished from its congeners by the combination of the following characteristics: (1) medium-sized body; (2) finger tips extending beyond the snout while forelimbs stretched forward; (3) tips of forelimbs and hindlimbs reaching and overlapping when folded towards the body; (4) snout edge round; (5) absence of small papillae in the male cloacal fissure; (6) dorsal ridges narrow, with width smaller than the eye diameter; (7) head length (HDL) greater than head width (HDW) (HDL/HDW = 1.04~1.22); (8) orange markings absence on the parotoid; (9) nodule-like warts on body sides continuous; (10) vomerine teeth not connected at the tip, extending to the occipital bone at the posterior end.

Communicator whistles: A Trek through the taxonomy of the Boophis marojezensis complex reveals seven new, morphologically cryptic treefrogs from Madagascar (Amphibia: Anura: Mantellidae)

Abstract The Malagasy stream-breeding treefrog species Boophis marojezensis contains bioacoustically and genetically highly divergent populations. Some of these populations have been defined as candidate species and emit somewhat bizarre advertisement calls consisting of multiple whistle-notes. We here enable a long-overdue taxonomic revision of this species complex by applying a museomics approach to sequence DNA from the holotype of B. marojezensis. Based on an integrative approach that combines divergence levels in mitochondrial DNA and in three nuclear-encoded genes, morphological data, and bioacoustic comparisons, we conclude that eight different species exist in this complex, seven of which are formally described herein as new. Although morphological differences between species are small and mainly separate small-sized from larger-sized species, conclusive evidence for the new species comes from their sympatric and sometimes syntopic occurrence without haplotype sharing in three nuclear genes and under maintenance of bioacoustic differences. Uncorrected genetic divergences in the mitochondrial 16S rRNA gene are >3% in almost all cases, and in some cases up to 8%. In reference to the otherworldly sounds by which these frogs fill Malagasy rainforests, some of them reminiscent of sounds of technical equipment in the fictional “Star Trek” universe, we here name and describe the seven new species in honor of fictional captains of starships, namely B. kirkisp. nov., B. picardisp. nov., B. siskoisp. nov., B. janewayaesp. nov., B. archerisp. nov., B. pikeisp. nov., and B. burnhamaesp. nov. The majority of these species occur in northern Madagascar, where up to three species can occur in immediate geographical proximity, e.g., B. marojezensis, B. burnhamaesp. nov. and B. pikeisp. nov. at different elevations in the Marojejy Massif. South of 16°S latitude, only B. janewayaesp. nov., B. picardisp. nov., and B. kirkisp. nov. are found, with the latter extending southwards to Ranomafana National Park. Our study confirms the existence of numerous morphologically cryptic and microendemic species among Madagascar’s amphibians, some of which are known only from unprotected sites and require adequate conservation management.

Revision of the Chinese termite genus Peribulbitermes (Blattodea, Isoptera, Termitidae): A new synonym of Ahmaditermes

The genus Peribulbitermes is a small genus in Nasutitermitinae comprising three species distributed only in China. Type specimens of Peribulbitermes dinghuensis Li, 1985 and Peribulbitermes jinghongensis Li, 1985 are obtained in our study. After examining the morphological characters of the specimens, it is found that P. jinghongensis cannot be differentiated from P. dinghuensis by the diagnostic characters reported in original description. We propose that P. jinghongensis is the junior synonym of P. dinghuensis. Additionally, molecular phylogenetic relationships of Peribulbitermes, with its morphologically similar genera, Bulbitermes, Ahmaditermes and Nasutitermes, as well as other nine genera in Nasutitermes Group of Nasutitermitinae were analysed based on mitochondrial COI, COII and 16S rRNA genes. The results show that the relationship between Peribulbitermes and Ahmaditermes is the closest. Peribulbitermes should be downgraded to synonymy of Ahmaditermes. P. dinghuensis and Peribulbitermes parafulvus (Tsai and Chen, 1963) should be transferred to Ahmaditermes as Ahmaditermes dinghuensis and Ahmaditermes parafulvus.

Improvement in Microbiota Recovery Using Cas-9 Digestion of Mānuka Plastid and Mitochondrial DNA

Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (mānuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.

Carcinoplaxmistio Ng & Mitra, 2019 (Crustacea, Decapoda, Goneplacidae): additional records and genetic differentiation of allied taxa.

The goneplacid crab, Carcinoplaxmistio Ng & Mitra, 2019, was originally described from West Bengal, India, in the northern Indian Ocean. Additional material of C.mistio from off Tamil Nadu in the southeast of India revealed a high degree of size-associated variation in the structures of the anterolateral tooth of the carapace, chelipeds, and male and female pleons. In addition to an in-depth morphological examination of C.mistio, this study also records the natural coloration of the species and conducts a genetic comparison (with mitochondrial COI and 16S rRNA genes) with its close relatives, C.haswelli (Miers, 1884) and C.purpurea Rathbun, 1914. Molecular comparison of C.mistio with its morphologically closest congener, C.haswelli from northern Australia and the western Pacific, corroborates their status as separate species. The genetic sequence of C.mistio, however, is similar to that of C.purpurea from the West Pacific, although these two species can easily be distinguished by distinct carapace and ambulatory leg characters. The present study provides some possible explanations for the genetic and morphological incongruence observed between C.mistio and C.purpurea and highlights the need for a detailed molecular study for Carcinoplax H. Milne Edwards, 1852, to appreciate the evolution of various morphological characters in the genus.

Fine-scale phylogeography of Tripneustes gratilla revealed a core-periphery pattern in the South China sea

The Core-Periphery Hypothesis (CPH) is a theoretical framework in phylogeography used to examine the spatial distribution of genetic diversity. The tropical Indian-Western Pacific (IWP), serving as a prominent marine biodiversity hotspot, constantly exports species and evolutionary novelties to peripheral habitats. Many species display genetic patterns consistent with CPH predictions from the IWP to periphery regions. However, the presence of CPH’s genetic signals in intermediate distribution zones remains underexplored. Here, we collected four sea urchin populations of Tripneustes gratilla in the South China Sea and analysed their population structure and evolutionary patterns using 13 morphological parameters, one mitochondrial 16S rRNA gene and nine polymorphic microsatellites. The results revealed substantial genetic homogeneity among the populations, implying a single origin from a glacial refugium. Nonetheless, morphological and microsatellite data distinctly differentiate the northernmost Fengjiawan population from the others, which could be attributed to increased environmental selection pressures such as temperature. Analysis of genetic diversity variation along latitudinal gradients and the relationship between geographic and genetic distances align with CPH expectations for both 16S and microsatellites, albeit to varying degrees of significance. This study enhances our understanding of tropical marine invertebrate evolution and supports the applicability of the CPH model at a fine-scale level.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding

ObjectiveLeptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. MethodsForty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. ResultsWe revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. ConclusionWe repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

Expanded range of Nasutitermes callimorphus Mathews, 1977 (Isoptera: Termitidae: Nasutitermitinae), comparison with N. corniger (Motschulsky, 1855) and N. ephratae (Holmgren, 1910), and synonymy of N. dasyopsis Thorne, 1989 into N. nigriceps (Haldeman, 1854)

The imago of N. callimorphus is described for the first time. Nasutitermes callimorphus occurs from Mexico to Paraguay. Nasutitermes callimorphus is smaller in all measurements but generally resembles its widely distributed sympatric congeners, N. corniger (Motschulsky, 1855) and N. ephratae (Holmgren, 1910). Molecular phylogenetic analysis of a portion of the mitochondrial 16S rRNA gene and the COII mtDNA marker revealed that N. callimorphus forms a distinct clade using both maximum parsimony and maximum likelihood analysis. Unlike N. corniger and N. ephratae, N. callimorphus does not build epigeal or arboreal carton nests. We further regard N. dasyopsis Thorne, 1989 as a junior synonym of N. nigriceps (Haldeman, 1854).

From ctenophores to scyphozoans: parasitic spillover of a burrowing sea anemone

Most host-parasite associations are explained by phylogenetically conservative capabilities for host utilization, and therefore parasite switches between distantly related hosts are rare. Here we report the first evidence of a parasitic spillover of the burrowing sea anemone Edwardsiella carnea from the invasive ctenophore Mnemiopsis leidyi to two scyphozoan hosts: the native Mediterranean barrel jellyfish Rhizostoma pulmo and the invasive Indo-Pacific nomad jellyfish Rhopilema nomadica, collected from the Eastern Mediterranean Sea. Edwardsiella carnea planulae found in these jellyfish were identified using molecular analyses of the mitochondrial 16S and nuclear 18S rRNA genes. Overall, 93 planulae were found on tentacles, oral arms, and inside of the gastrovascular canals of the scyphomedusae, whereas no infection was observed in co-occurring ctenophores. DNA metabarcoding approach indicated seasonal presence of Edwardsiella sp. in the Eastern Mediterranean mesozooplankton, coinciding with jellyfish blooms in the region. Our findings suggest a non-specific parasitic relationship between Edwardsiella carnea and various gelatinous hosts based on shared functionality rather than evolutionary history, potentially driven by shifts in host availability due to jellyfish blooms. This spillover raises questions about the ecological impacts of parasitism on native and invasive scyphozoan hosts and the potential role of Edwardsiella in controlling their populations.

First Evidence of Rickettsia conorii Infection in Dogs in Northern Tunisia.

A cross-sectional study was carried out, between April 2021 and June 2022, to understand the role of dogs in the circulation of rickettsiosis in Tunisia. The presence of specific IgG antibodies against Rickettsia conorii was analyzed by indirect immunofluorescence test. By qPCR, blood and ticks were collected from 136 dogs examined at the Canine Department of National School for Veterinary Medicine of Tunisia. These dogs were also analyzed to detect Rickettsia DNA. The rate of Rickettsia seropositivity in 136 dogs was 55.14%. A total of 51 (53%) seropositive dogs showed clinical and biological signs such as fever and anorexia as well as thrombocytopenia and anemia. By qPCR, targeting the mitochondrial 16S rRNA gene, no Rickettsia DNA was detected in the blood. On the other hand, qPCR followed by sequencing revealed the presence of R. conorii subsp. raoultii in 7 tick pools of the 51 pools composed of the 227 ticks collected. A One Health approach to raise the awareness of dog owners to control tick infestations is imperative, given the dangers of canine zoonoses.

A new species of colorful Platypelis (Amphibia: Microhylidae) from the Tsaratanàna and Bemanevika massifs in northern Madagascar

Frogs of the genus Platypelis are known to have their center of species richness in the mountain massifs of northern Madagascar. We here formally describe a new species of Platypelis from this region. Platypelis saikamavo sp. nov. has been known for over 20 years but its taxonomic identity has only recently been robustly clarified as a result of ongoing taxonomic revisions of other Platypelis species. The new species is known from rainforest on the Tsaratanàna Massif and Bemanevika, at elevations of 1000–1530 m above sea level. It is characterized by an isolated phylogenetic position and is genetically highly divergent, with an uncorrected pairwise distance ≥4.5% in the mitochondrial 16S rRNA gene to all other nominal species of Platypelis. It has a unique combination of morphological and chromatic character states such as a relatively large body size of 28–31 mm in snout–vent length, distinct superciliary tubercles, and bright yellow color around the forelimb insertion and on the posterior abdomen where this color extends in the form of flash marks onto the inguinal region.

Further evidence for diversification within the Diogenes pugilator complex (Anomura, Diogenidae) in the Mediterranean and Black Seas

Abstract We document an unrecognised genetic lineage within the European hermit crab Diogenes pugilator species complex. Specifically, we propose the revalidation of the previously synonymised species D. ponticus, for specimens previously identified as D. pugilator from the Adriatic, Ionian, Aegean, and Black Seas. Analyses of the mitochondrial genes COI and 16S rRNA resulted in a clear genetic separation to East Atlantic and Western Mediterranean populations of Diogenes. Morphological analyses confirmed differences in the individuals assigned to D. ponticus compared to the congeners D. curvimanus and D. pugilator and thereby a concordance between molecular and morphological characters. Despite the relatively high morphological resemblance to D. armatus, the molecular differentiation and the remarkable geographic segregation within the species complex suggest at least one speciation or radiation event in the past, underlining the growing importance of molecular methods in the identification of species and evolutionarily significant units. Major oceanographic processes that are known to shape genetic variability and population genetic structure in the marine environment are pointed out as possible structuring factors for the observed intrageneric divergence. The evidence of long evolutionary independence is therefore considered sufficient to re-establish the validity of D. ponticus as a valid species in the northern-central and north-eastern Mediterranean Sea and the Black Sea and is redescribed accordingly.

Population structure and haplotype network analyses of Hyalomma anatolicum based on the large subunit ribosomal RNA (16S rRNA) gene.

Hyalomma anatolicum, an Anatolian hard tick is a well-recognized vector involved in the transmission of various pathogens to animals and humans. The present study elucidated the population structure and haplotype network of H. anatolicum based on the mitochondrial large subunit ribosomal RNA (16S rRNA) gene sequence. The population structure and haplotype network analysis of 75 sequences archived in the GenBank, including the 15 sequences generated herein, yielded 24 haplotypes. Haplotype 1 (Hap_1) was the predominant haplotype consisting of 45 sequences from India, China, Pakistan, Turkey, Egypt, Iraq, and Tajikistan. The complete haplotype network exhibited a stellate conformation, highlighting a recent population expansion. The overall dataset, together with the sequences corresponding to India, China, and Pakistan, showed a high haplotype (0.638 ± 0.065, 0.671 ± 0.103, 0.753 ± 0.099, and 0.854 ± 0.061, respectively) and low nucleotide (0.00407 ± 0.00090, 0.00525 ± 0.00196, 0.00680 ± 0.00233, and 0.00453 ± 0.00056, respectively) diversity, further emphasized a recent population expansion. The neutrality indices including Tajima's D, Fu and Li's D, and Fu and Li's F for the complete dataset (- 2.661, - 6.008, and - 5.649, respectively) as well as for the sequences from India (- 2.223, - 3.414, and - 3.567, respectively) were negative, suggesting deviation from neutrality and a recent population expansion. The present study provided novel insights into the population structure and haplotype networks of H. anatolicum based on the mitochondrial 16S rRNA gene, and the different tests inferred a low genetic differentiation and suggested a recent population expansion of this economically important tick species.

A new species of green pitviper of the Trimeresurus macrops complex (Reptilia: Serpentes: Viperidae) from South Central Coastal Region of Vietnam

We describe a new species of green pitviper from southern and central parts of coastal Vietnam based on morphological and molecular (2406 bp from cyt b, ND4, and 16S rRNA mitochondrial DNA genes) lines of evidence. Trimeresurus cyanolabris sp. nov. is distinguished from its congeners by the combination of the following morphological characters: small size, maximum known SVL of 638 mm; dorsal scales in 21 (rarely 23)–21–15 rows, moderately keeled except the outermost rows; ventral scales 166–178; subcaudal scales 52–75, all paired; hemipenis forked, calyculate, reaching the 8th subcaudal; eye bright yellow in both sexes; dorsal surface deep green lacking cross-bands; postocular white stripe missing in both sexes; ventrolateral stripe faintly present on the first few dorsal scale rows in males, absent in females; throat, chin, and lower labials in shades of blue. The new species forms a distinct clade on the phylogenetic tree of the genus Trimeresurus and differs from the morphologically similar T. rubeus by a significant divergence in cytochrome b mitochondrial DNA gene sequences (p = 6.0%). The new species is currently known from low- to mid-elevations (ca. 90–400 m a.s.l.) in tropical forests of central-southern Vietnam. This discovery further underlines the importance of this area as a local center of herpetofaunal diversity and endemism, which is under great threat of deforestation.

Larval morphology, vocalisations, and phylogenetic position of Crossodactylus dantei (Anura: Hylodidae): new insights into a little-known anuran species

Aspects of the biology and natural history of the endemic anuran Crossodactylus dantei remain unknown even almost 30 years since its description. In this study, we describe its larval stage, along with the advertisement and territorial calls, and assess its phylogenetic positioning based on a fragment of the 16S rRNA mitochondrial gene. The larval stage of C. dantei follows the morphological pattern of Hylodidae, corroborating the relatively uniform larval morphology of the family. However, it is still possible to observe characters that distinguish C. dantei from other species in the genus, such as the elliptical body in dorsal view, nostrils located closer to the snout than to the eyes, an elongated pre-nasal arena, and the absence of taenia tecti medialis and taenia tecti transversalis in the chondrocranium. Its advertisement call is characterised by a sequence of about 23 pulsed notes, with ascending amplitude modulation, and has a mean duration of 2.5 seconds, with a dominant frequency of 3356 Hz. The territorial call has a mean duration of 23 seconds, a dominant frequency of 3160 Hz, and two distinct types of notes. Phylogenetic analysis indicated the existence of two main lineages in the genus. Crossodactylus dantei was recovered with high statistical support as a sister species to the clade composed of C. gaudichaudii and C. timbuhy. Furthermore, the group formed by these three species was recovered as the sister lineage of C. trachystomus. In summary, the information presented here represents a significant advance in the knowledge of this enigmatic and rare species of the northern Atlantic Forest, providing baseline data for future ecological and evolutionary studies of this unique frog. Keywords: tadpole, oral anatomy, chondrocranium, calls, phylogeny, Atlantic Forest

Lebbeus dentatus n. sp. (Decapoda, Thoridae) described from a seamount of the Caroline Plate, NW Pacific

Based on the female specimen collected from an unnamed seamount on the Caroline Plate in the NW Pacific, a new species of the thorid genus Lebbeus White, 1847, L. dentatus n. sp., is described and illustrated. The new species belongs to a species group characterized by the possession of epipods on the pereopods 1 and 2, but is characteristic in the carapace having a strong supraorbital tooth extending to 0.3 length of the rostrum and largely unarmed dorsal margin of the rostrum. Integrative approach using morphological comparison and genetic analyses with the mitochondrial COI and 16S rRNA genes is taken to clarify the specific identities of the new taxon. An identification key to species belonging to the species group of Lebbeus characterized by the possession of epipods on the pereopods 1 and 2 is presented.

Comparative Cytogenetics of the Malagasy Ground Geckos of the Paroedura bastardi and Paroedura picta Species Groups.

We present a comparative chromosome study of several taxa of the Malagasy ground geckos of the Paroedura bastardi and P. picta species groups. We employed a preliminary molecular analysis using a trait of the mitochondrial 16S rRNA gene (of about 570 bp) to assess the taxonomic status of the samples studied and a cytogenetic analysis with standard karyotyping (5% Giemsa solution), silver staining (Ag-NOR staining) and sequential C-banding (C-banding + Giemsa and + fluorochromes). Our results show that all the taxa studied of the P. bastardi group (P. ibityensis, P. rennerae and P. cf. guibeae) have a similar karyotype composed of 2n = 34 chromosomes, with two metacentric pairs (1 and 3) and all other pairs being acrocentric. Chromosome diversification in the P. bastardi group was mainly linked to the diversification of heteromorphic sex chromosome systems (ZZ/ZW) in P. ibityensis and P. rennerae, while no heteromorphic sex chromosome pair was found in P. cf. guibeae. The two taxa investigated of the P. picta species group (here named P. picta and P. cf. picta based on molecular data) showed the same chromosome number of 2n = 36, mostly acrocentric elements, but differed in the number of metacentric elements, probably as a result of an inversion at chromosome pair 2. We highlight that the genus Paroedura is characterized by the independent diversification of heterogametic sex chromosomes in different evolutionary lineages and, similarly to other phylogenetically related gecko genera, by a progressive formation of a biarmed element by means of tandem fusions and inversions of distinct pairs.

Mitochondrial DNA evidence reflects high genetic divergence of Amynthas aspergillum (Oligochaeta: Megascolecidae) in southern China.

Amynthas aspergillum (Perrier, 1872), a natural resource used in traditional Chinese medicine (Guang-dilong) with high economic value, is widely distributed in forests and farmland habitats in the hilly areas of southern China. To investigate the extent of genetic differentiation and diversity in A. aspergillum, a population genetic structure study was performed on 157 samples from 75 locations in southern China using the mitochondrial genes COI, COII, 12S rRNA, 16S rRNA, and NDI. The results indicated that A. aspergillum had a high level of genetic diversity, and variation within populations was the main source of the total variation. Six deeply divergent mitochondrial clades (I-VI) were detected using both phylogenetic tree and haplotype network analyses. This finding was supported by the high Kimura two-parameter genetic distance and the pairwise fixation index value obtained based on the COI gene. No significant phylogeographic structures were observed. The widespread geographic distribution of clades II, IV, and VI suggested a recent demographic expansion based on multiple analysis results. These results include a high level of Hd and low π, star-shaped haplotype network structures with a high number of less frequent haplotypes, significantly negative neutrality test values, and a unimodal mismatch distribution pattern. The divergence time estimates and reconstruction of the ancestral area revealed that A. aspergillum originated in Guangxi Province and underwent initial intraspecific diversification in the early Pliocene to generate clade I. Then, it gradually dispersed eastward and rapidly differentiated into clades II-V during the Pleistocene. The Yunnan-Guizhou Plateau and Nanling and Wuyi Mountains might act as geographical barriers for the spread of A. aspergillum to the west and north.

Next generation sequencing improves the resolution of detecting mixed host blood meal sources in field collected arboviral mosquito vectors.

Accurate knowledge of blood meal hosts of different mosquito species is critical for identifying potential vectors and establishing the risk of pathogen transmission. We compared the performance of Miseq next generation sequencing approach relative to conventional Sanger sequencing approach in identification of mosquito blood meals using genetic markers targeting the 12S rRNA and cytochrome oxidase I (COI) genes. We analysed the blood meals of three mosquito vector species (Aedes aegypti, Aedes simpsoni s.l. and Culex pipiens s.l.) collected outdoors, and compared the frequency of single- versus multiple-blood feeding. Single host blood meals were mostly recovered for Sanger-based sequencing of the mitochondrial 12S rRNA gene, whereas Miseq sequencing employing this marker and the COI marker detected both single and multiple blood meal hosts in individual mosquitoes. Multiple blood meals (two or more hosts) which mostly included humans were detected in 19%-22.7% of Ae. aegypti samples. Most single host blood meals for this mosquito species were from humans (47.7%-57.1%) and dogs (9.1%-19.0%), with livestock, reptile and rodent hosts collectively accounting for 4.7%-28.9% of single host blood meals. The frequency of two or more host blood meals in Ae. simpsoni s.l. was 26.3%-45.5% mostly including humans, while single host blood meals were predominantly from humans (31.8%-47.4%) with representation of rodent, reptile and livestock blood meals (18.2%-68.2%). Single host blood meals from Cx. pipiens s.l. were mostly from humans (27.0%-39.4%) and cows (11.5%-27.36%). Multiple blood meal hosts that mostly included humans occurred in 21.2%-24.4% of Cx. pipiens s.l. samples. Estimated human blood indices ranged from 53%-76% for Ae. aegypti, 32%-82% for Ae. simpsoni s.l. and 26%-61% for Cx. pipiens s.l. and were consistently lower for Sanger-based sequencing approach compared to Miseq-based sequencing approach. These findings demonstrate that Miseq sequencing approach is superior to Sanger sequencing approach as it can reliably identify mixed host blood meals in a single mosquito, improving our ability to understand the transmission dynamics of mosquito-borne pathogens.

An innovative molecular approach towards the cost-effective entomological authentication of honey

Honey authentication and traceability are crucial not only for economic purposes but also for ensuring safety. However, the widespread adoption of cutting-edge technologies in practical applications has been hampered by complex, time-consuming sample pre-treatment processes, the need for skilled personnel, and substantial associated expenses. This study aimed to develop a simple and cost-effective molecular technique to verify the entomological source of honey. By utilizing newly designed primers, we successfully amplified the mitochondrial 16S ribosomal RNA gene of honey bees from honey, confirming the high quality of the extracted DNA. Employing RFLP analysis with AseI endonuclease, species-specific restriction patterns were generated for honey derived from six closely related honey bees of the Apis genus. Remarkably, this method was proven equally effective in identifying heat-treated and aged honey by presenting the same RFLP profiles as raw honey. As far as we know, this is the initial research of the simultaneous differentiation of honey from closely related honey bee species using the restriction endonuclease AseI and mitochondrial 16S rRNA gene fragments. As a result, it holds tremendous potential as a standardized guideline for regulatory agencies to ascertain the insect origins of honey and achieve comprehensive traceability.