Species Of Pheretima Research Articles

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics

Pheretima, also called “earthworms”, is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines (CPMs) in Chinese Pharmacopoeia (2020 edition). However, its zoological origin is unclear, both in the herbal market and CPMs. In this study, a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms (restricted search, open search, and de novo) was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima, including Pheretima aspergillum (PA), Pheretima vulgaris (PV), and Metaphire magna (MM). We identified 10,477 natural peptides in the PA, 7,451 in PV, and 5,896 in MM samples. Five specific signature peptides were screened and then validated using synthetic peptides; these demonstrated robust specificity for the authentication of PA, PV, and MM. Finally, all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills, revealing the inconsistent Pheretima species used in these CPMs. In conclusion, our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines, especially non-model species with poorly annotated protein databases.

A novel strategy to identify the species-specific peptide biomarkers in Pheretima aspergillum (E. Perrier) based on enzymatic digestion followed by LC-MS/MS methods

Guang Dilong [P. aspergillum (E. Perrier)], is an animal-derived traditional Chinese medicine made from the dried body of Pheretima aspergillum (E. Perrier) (TCM). Due to its widely application and high medical values, preparations of P. aspergillum (E. Perrier) may be adulterated by four other species, including three crucial Pheretima species [P. vulgaris (Chen), P. pectinifera (Mkhaeken), and P. guillemi (Michaelsen)] and one considerable adulteration [Metaphire magna (Chen)]. This study developed a novel and effective strategy for analyzing and authenticating Guang Dilong based on enzymatic digestion of protein. The nanoLC-MS/MS technique used to evaluate complete peptidomics profiles of trypsin-digested samples, resulting in the identification of species-specific peptide biomarkers in P. aspergillum (E. Perrier). The significance of different samples and peptides in the target species set was then investigated using mathematical set theory. Consequently, seven peptides were chosen as prospective biomarkers. Finally, five specific peptide biomarkers for differentiating Guang Dilong with other species were confirmed and validated using UFLC-MS/MS and MRM mode. The suggested technique may also be beneficial in evaluating the quality of other animal-derived goods for safety issues in order to avoid misidentification.

Earthworm diversity and populations in different habitats of Rajah Sikatuna Protected Landscape, Bohol, Philippines

An earthworm survey was conducted to determine the species composition, diversity, and species richness in the grassland, agricultural areas, and secondary forest habitats of Rajah Sikatuna Protected Landscape (RSPL). Six indigenous morphospecies of earthworms were identified, which are putatively new species. These include one Amynthas species, three Polypheretima species, and two Pheretima species. Also, Pontoscolex corethrurus, an invasive species was found to be the most abundant species of earthworm in grassland and agricultural areas but not in the secondary forest. The diversity index showed a high level in the secondary forest with low species richness. Agricultural and grassland areas showed a low level of diversity. Land use and abiotic factors can be related to the composition of earthworm communities in different habitats. Further studies must be conducted for the conservation and sustainable management of its biodiversity and habitat.

A rapid and simple signature peptides-based method for species authentication of three main commercial Pheretima

Pheretima with various activities is a commonly used animal-derived traditional medicine in Asia countries. However, almost half of them are non-pharmacopoeia species in the market due to the similar morphological characteristics between medicinal and non-medicinal species. This study aims to establish an effective method based on signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna). Firstly, the species of 52 batches of commercial Pheretima were authenticated based on DNA barcodes. Secondly, proteomic analysis was performed for protein characterization of three main commercial Pheretima. Furthermore, their signature peptides were screened and validated using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, a simplified sample processing method was developed. Finally, large quantities of commercial Pheretima samples were analyzed for further verifying the feasibility of the signature peptides-based method. The result showed that the established method had a great application potential for authenticity identification of commercial Pheretima. SignificanceThe authenticity assessment of medicinal materials is a main issue in the quality control process as deceptive practices could imply severe health risks. In this study, a rapid and simple method based on signature peptides was established for species authentication of three main commercial Pheretima, which can be an effective alternative to complex DNA barcoding and difficult morphological identification, and provided a reference for improvement of Pheretima quality standards.

Authentication of three main commercial Pheretima based on amino acids analysis.

Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.

Marker peptide screening and species-specific authentication of Pheretima using proteomics.

Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.

Efficacy of Pheretima species for accumulation of heavy metal contaminants

Efficacy of Pheretima species for accumulation of heavy metal contaminants

First record of the earthworm genus Pheretima Kinberg, 1867 sensu stricto in Vietnam, with description of a new species (Annelida: Clitellata: Megascolecidae).

The earthworm genus Pheretima Kinberg, 1867 sensu stricto is recorded in Vietnam for the first time with a new species, P. vungtauensis sp. nov. The new species is characterized by three pairs of spermathecal pores in intersegments 6/7/8/9, spermathecal ducts with dense micronephridia, no genital markings in spermathecal and male regions, male pores located inside copulatory pouches, intestinal origin at xv, caeca simple from xxvii, and holandry. DNA-COI barcode sequences of the holotype are most similar to those of Metaphire houlleti (Perrier, 1872) from Thailand and not to other species of Pheretima.

Food Preference of Endemic Sri Lanka Yellow-eared Bulbul (Pycnonotus penicillatus) in Breeding and Non-breeding seasons at Horton Plains National Park

Food preference of Sri Lanka Yellow-eared Bulbul (Pycnonotus penicillatus) in Breeding and non-breeding seasons was studied at the montane cloud forests of Horton Plains National Park, situated in the highland plateau of the central highlands, from September 2015 to July 2017 in three consecutive days per month. P. penicillatus is an endemic near threatened species. They have two breeding seasons; February-May and August-October. Direct foraging observations and faecal sample analysis methods were used to collect data. Four mist nets (3x6 m2/25 mm mesh size) were allocated from 0600 to 1800 h in mist netting days. Droppings of the bird was later scraped individually into collecting bottles with 70% alcohol and stored. Some droppings were collected in the field. These samples were taken to the laboratory for further analysis. The samples were first divided into two main parts as high plant matter samples and high insect matter samples by visually observing the faecal matter. ‘High plant matter’ faecal samples were directly analysed. High insect matter samples were analysed after using standard protocols. The seeds of the plants were identified by the keys and, insect parts were identified to order level by using insect identification guides. Within the study period there were 1,903 field observations and 50 faecal sample analysis were performed. In the breeding season 87.9% of the feeding observations were consisted with 18 species of plants and remain 12.1% was consisted with animal parts from one Pheretima species, thirteen species of insects, including three species from Order Diptera, three species from Order Lepidoptera, two species from Order Coleoptera, three species from Order Hymenoptera, one species from Order Blattodea and one species from Order Hemiptera. In the non-breeding season 98.1% of the feeding observations were consisted with 15 species of plants and other 1.9% was completed with four species of animal parts, including one species from Order Diptera, one species from Order Lepidoptera and two species from Order Coleoptera. Present study revealed that P. penicillatus preferred more animal food items in the breeding season, possibly to secure the extra energy requirements during their breeding season. These studies will be important for conservation of P. penicillatus, by using species-specific conservation plans in the future.Keywords: Sri Lanka Yellow-eared Bulbul, Endemic birds, Horton Plains National Park, Food preference, Breeding and non-breeding seasons

New species of Pheretima, Amynthas, Polypheretima, and Pithemera (Clitellata: Megascolecidae) from Mindanao and Associated Islands, Philippines.

Nonillon M. Aspe and Samuel W. James (2016) Studies over the past decade have shown the Philippine native earthworm fauna to be highly diverse, with apparent high endemism among islands and localities. Here we describe 17 new species of pheretimoid earthworms collected from priority conservation sites on Mindanao and associated islands. Nine species of the subgenus Pheretima (Pheretima), distinguished by the absence of secretory diverticula on the coelomic surface of the copulatory bursae, were detected. Pheretima acia n. sp., P. dinagatensis n. sp., P. enormis n. sp., P. hamiguitanensis n. sp., P. lantapanensis n. sp. belong to the P. urceolata species group, with a pair of spermathecal pores in 5/6; P. timpoongensis n. sp., P. camiguinensis n. sp., P. sibucalensis n. sp., and P. apoensis n. sp. belong to the P. sangirensis group, with a pair of spermathecal pores in 7/8. Pheretima (Parapheretima) pandanensis n. sp. and P. (Parapheretima) boaensis n. sp., members of the subgenus Pheretima (Parapheretima), with secretory diverticula on the coelomic surface of the copulatory bursae, are the first Philippine records of Parapheretima. In the genus Amynthas, A. dinagatensis n. sp. is athecate; A. cagdianaoensis n. sp. belongs to the belongs to the A. supuensis species group, with a pair of spermathecal pores at 8/9; and A. talaandigensis n. sp. belongs to A. rimosus group, with intrasegmental spermathecae in 6, 7, 8, and 9. In the genus Polypheretima, P. bukidnonensis n. sp. belongs to the Po. elongata species group, with paired genital markings on xix and successive segments, and paired spermathecal batteries in vi and/or vii; Po. zamboangensis n. sp., either has spermathecal pores in 5/6/7 or lacks them, and has a metandric male sexual system. Pithemera nolani n. sp. is unique among congeners in having only one pair of spermathecal pores in 5/6 and in having copulatory bursae. The geological history of Mindanao, which is a collision complex between three arc systems, may have contributed to the high diversity of the earthworm fauna on Mindanao. Future molecular studies can help elucidate the evolutionary relationships and geographical distribution of species and populations, and shed light on mechanisms of earthworm diversification in the Philippines.

New species of Pheretima (Oligochaeta: Megascolecidae) from the Mt. Malindang Range, Mindanao Island, Philippines

We provide descriptions, with illustrations of internal structures, for 18 new species of Pheretima from Mt. Malindang, Misamis Occidental Province, Mindanao Island, Philippines. Among the 18 species, 11 belong to the P. sangirensis species group, characterized by having a pair of spermathecal pores in the intersegmental furrow of 7/8 and lacking penial sheaths in the copulatory bursae: P. maculodorsalis n. sp., P. tigris n. sp., P. immanis n. sp., P. lago n. sp., P. nunezae n. sp., P. boniaoi n. sp., P. malindangensis n. sp., P. misamisensis n. sp., P. wati n. sp., P. longiprostata n. sp., and P. nolani n. sp. One species, P. longigula n. sp., belongs to the P. montana species group, characterized by having a pair of spermathecal pores in the intersegmental furrow of 7/8 and penial sheaths in the copulatory bursae. Two species, P. vergrandis n. sp. and P. concepcionensis n. sp., are monothecal. Three species, P. adevai n. sp., P. lluchi n. sp., and P. potonganensis n. sp., belong to the P. darnleiensis species group, characterized by having either four or five pairs of spermathecae from vi to ix, with a fifth pair variably present in segment v. One species, P. subanensis n. sp., is athecate. All species described here from the Mt. Malindang Range are probably native rather than introduced, and probably do not represent range extensions of species known from neighboring islands in Southeast Asia. We provide an identification key to the Pheretima species from Mt. Malindang.

Testing on three determining methods of genetic diversity on earthworm species belonging to the Pheretima species group in the Mekong delta

Testing on three determining methods of genetic diversity on earthworm species belonging to the Pheretima species group in the Mekong delta

New Species Of Pheretima, Pithemera, And Polypheretima (Clitellata: Megascolecidae) From Kalbaryo, Luzon Island, Philippines

Eight new species of pheretimoid earthworms collected from Kalbaryo, Ilocos Norte Province, Luzon Island, the Philippines are described herein: four new species of Pheretima, three new species of Pithemera, and one new species of Polypheretima. All the Pheretima species lack genital markings. Pheretima margaritata, new species, Pheretima kalbaryoensis, new species, and Pheretima thaii, new species, have spermathecae in vi-ix, but Pheretima globosa, new species, has spermathecae in vii-ix. The distances between the male pores in Pheretima margaritata, new species, Pheretima kalbaryoensis, new species, Pheretima thaii, new species, and Pheretima globosa, new species, are 2.4 mm, 4.5 mm, 1.4 mm, and 1.7 mm, respectively. Pithemera glandis, new species, and Pithemera fusiformis, new species, have fi ve pairs of spermathecae in v-ix. Pithemera levii, new species, has four pairs in v-viii. Pithemera glandis, new species, has no genital markings, but Pithemera fusiformis, new species, has paired genital markings in xix and xx. Pithemera levii, new species, has paired genital markings in xviii and xix. Polypheretima pagudpudensis, new species, has spermathecae in vi-vii, and genital markings paired in xvii, xix, and xx. Descriptions of the new species are provided, including illustrations of the ventral view, male pore region, and spermathecae.

New earthworms of the Pheretima urceolata species group (Oligochaeta: Megascolecidae) from southern Luzon, Philippines

Earthworm specimens were collected from the Bicol region, southern Luzon Island, the Philippines. Seven species of the genus Pheretima Kinberg, 1867 are described from Bicol: Pheretima bicolensis sp. nov., Pheretima viracensis sp. nov., Pheretima camarinensis sp. nov., Pheretima doriae sp. nov., Pheretima batoensis sp. nov., Pheretima buhiensis sp. nov., and Pheretima gorasi sp. nov. The seven new Pheretima species all key to the urceolata group in Sims & Easton (1972) with a single pair of spermathecal pores in 5/6. Pheretima bicolensis sp. nov. has spermathecal pores 0.05–0.06 circumference apart, copulatory bursae openings 0.14 circumference apart. Pheretima viracensis sp. nov., has spermathecal pores 0.32–0.36 circumference apart; copulatory bursae openings 0.20 circumference apart. Pheretima camarinensis sp. nov., has spermathecal pores 0.18–0.20 circumference apart; copulatory bursae openings 0.19–0.20 circumference apart. Pheretima doriae sp. nov., has spermathecal pores 0.32 circumference apart; copulatory bursae openings 0.32 circumference apart. Pheretima batoensis sp. nov., has spermathecal pores 0.21–0.23 circumference apart; copulatory bursae openings 0.15–0.18 circumference apart. Pheretima buhiensis sp. nov., has spermathecal pores 0.04–0.06 circumference apart; copulatory bursae openings 0.07–0.12 circumference apart. Pheretima gorasi sp. nov., has spermathecal pores 0.06–0.07 circumference apart; copulatory bursae openings 0.10 circumference apart. Descriptions of the new species are provided, including illustrations of the ventral view, and spermathecae.

Two new earthworms of the genus Pheretima (Oligochaeta: Megascolecidae) from Mt. Isarog, Luzon Island, Philippines

Earthworm specimens were collected from Mt. Isarog, Camarines Sur Province, the Philippines. Two new species of the genus Pheretima are described from Mt. Isarog: Pheretima abiadai sp. nov. and Pheretima nagaensis sp. nov. The two new Pheretima species key to the urceolata group with a single pair of spermathecal pores in 5/6. Pheretima abiadai sp. nov. has spermathecal pores 0.25 circumference apart, copulatory bursae openings 0.19 circumference apart. Pheretima nagaensis sp. nov. has spermathecal pores 0.29 circumference apart; copulatory bursae openings 0.25 circumference apart. Descriptions of the new species are provided, including illustrations of the ventral view, male pore region, and spermathecae.

New earthworms of Pheretima and Pithemera (Oligochaeta: Megascolecidae) from Mt. Arayat, Luzon Island, Philippines

Three new species of Pheretima and two new species of Pithemera are described from Mt. Arayat: Pheretima arayatensis sp. n., Pheretima simsi sp. n., and Pheretima castilloi sp. n., Pithemera rotunda sp. n., and Pithemera philippinensis sp. n. Pheretima arayatensis sp. n., Pheretima simsi sp. n., and Pheretima castilloi sp. n. have spermathecae in segment ix, vi, and vi-viii, respectively. Pithemera rotunda sp. n., and Pithemera philippinensis sp. n. have spermathecae in segment v-ix. The former species has numerous circular genital markings, but the latter Pithemera species has no genital markings. Descriptions of the new species are provided, including illustrations of the ventral view, male pore region, and spermathecae.

A Comparative Study on Earthworm Hemoglobins: An Amino Acid Sequence Comparison of Monomer Globin Chains of Two Species, Pontodrilus matsushimensis and Pheretima communissima That Belong to the Family Megascolecidae

The monomer subunits of giant extracellular hemoglobins from earthworms Pontodrilus matsushimensis and Pheretima communissima that belong to the family Megascolecidae, Oligochaeta, were purified by a reversed-phase column, Resource RPC, and sequenced. The complete amino acid sequences of the two monomeric globin chains were determined: 141 amino acid residues with a molecular weight of 16,366 Da for Pontodrilus matsushimensis and 140 amino acid residues with a molecular weight of 16,000 for Pheretima communissima, respectively. The Pontodrilus matsushimensis monomer globin has three cysteine residues, and the two located at positions 2 and 131 are conserved as those observed in all annelids and contribute to form a disulfide-bonded interchain. The third cysteine residue at position 73 is the first evidence for the annelid monomer globin subunits. The physiological functions of the third cysteine residue, however, are still unknown. The monomer sequences of the two species were aligned with those of five known sequences from annelids, including a polychaete, Tylorrhynchus heterochaetus, and four oligochaetes, Pheretima hilgendorfi, Pheretima sieboldi, Lumbricus terrestris and Tubifex tubifex. Using computer analysis, a 87.9% identity of the amino acid sequences between two monomeric subunits of Pheretima communissima and Pheretima hilgendorfi hemoglobins showed the highest degree of sequence similarity. A molecular phylogenetic tree of seven species of annelids has constructed, suggesting that the divergence times among the three species of Pheretima and between Pheretima and Pontodrilus were 50 to 100 and about 209 million years ago, respectively.

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of the earthworm, Pheretima hilgendorfi.

The complete amino acid sequence of the monomer subunit of Pheretima hilgendorfi hemoglobin was determined: It consists of 140 amino acid residues, including a disulfide bond but no methionine, and has a molecular weight of 16,107 Da. Using computed analyses (amino acid maximum homology) with known sequences of monomer subunits of earthworm's hemoglobins, 115 (82%) were found to be identical with those in the corresponding positions of chain I (monomer subunit) of Pheretima sieboldi hemoglobin; 81 residues (55%), 71 residues (47%), and 66 residues (43%) were found to be in identical positions of the sequences of chain I of Lumbricus terrestris hemoglobin, chain I of Tubifex tubifex hemoglobin and chain I of Tylorrhynchus heterochaetus hemoglobin. Orthologous sequence data of monomer globins that belong to the strain A of annelid hemoglobins are discussed as useful clues for investigation of the divergence pattern of Pheretima species.

Studies on a new oxalate-decomposing bacterium, Pseudomonas oxalaticus.

Since Bhat and Barker (1948) reviewed the existing literature and reported on the isolation and characteristics of an oxalate-decomposing Vibrio, only two attempts appear to have been made to study the problem of oxalate decomposition by microorganisms. One of the studied reported (Janota, 1950) relates to the utilization of oxalate by Psudomonas extorquens Basalik, whereas the other by Muiller (1950) pertains to the oxalate-decomposing species of Proactinomyces isolated from soils, water, and sheep rumina. The purpose of this paper is to present the details of the isolation and the description of another oxalate-decomposing bacterium isolated from the intestinal tract of common Indian earthworms including the Pheretima species.

SCIENCE IN WESTERN SZECHUAN

II. BIOLOGICAL AND SOCIAL SCIENCES IN the preceding article* something has been said of the state of physics and chemistry in the five universities at the great city of west Szechuan, Chengtu. On the whole, the biological work there is even more active. On the West China Union (Huahsi) University campus some excellent taxonomic and ecological work is being done. Thus Dr. Chang Ming-Chun of Huahsi specializes on gastropod systematics and embryology, while Dr. Liu Cheng-Chao of Yenching is a really outstanding authority on Asiatic Amphibia. His collections and data are astonishing, including torrent adaptations, strange breeding habits, etc., and many new species, such as Vibrissaphora boringii, a whiskered frog. There are great opportunities for experimental embryology here, which have so far not been taken advantage of. Dr. Liu himself goes on collecting expeditions in the Tibetan borderlands which involve real hardship and danger. For comparative physiology also there are great opportunities, for example, to work with the giant earthworms of the Mount Omei region, species of Pheretima, commonly up to 3 ft. long and at least 1 in. in diameter.