A new species of guizhou macropes, its dna standard detection gene and application

Abstract

The application belongs to the technical field of new species gene detection of insects, and particularly relates to a Guizhou large-mouthed worm new species, a DNA standard detection gene thereof and application. The Guizhou large-mouthed worm is separated from a freshwater environment in Guizhou province of China, the worm can actively prey on Rhabditis sp. nematodes, and the Guizhou large-mouthed worm is identified by combining traditional morphological identification and molecular classification methods, so that the accuracy of species identification can be ensured.

Description

Technical Field

[0001] This invention belongs to the field of new insect species gene detection technology, specifically involving a new species of Guizhou largemouth flatworm, its DNA standard detection gene and its application. Background Technology

[0002] Nematodes are a significant group of pests in agriculture, forestry, and aquaculture, especially root-knot nematodes and saprophytic nematodes, which cause severe losses to crop yield and quality. Currently, nematode control mainly relies on chemical nematicides, but long-term use easily leads to increased nematode resistance, environmental pollution, and agricultural product residues, among other problems. Therefore, the development of safe, efficient, and environmentally friendly biological control resources has become a research hotspot in this field.

[0003] Planarians (Platyhelminthes: Rhabditophora) are a group of lower, bilaterally symmetrical animals widely distributed in freshwater, seawater, and moist soil environments worldwide. The genus *Macrostomum* is particularly diverse, with over 200 species reported globally. Current research on planarians primarily focuses on their morphology, taxonomy, developmental biology, and reproductive mechanisms. While some planarians (such as *Trigonella triangularis* and *Platyhelminthes monogastropoda*) have been reported to prey on mosquito larvae or soil nematodes, there are no systematic reports of *Macrostomum* species preying on nematodes, and no patents or technologies have been found for their use in nematode biological control. Summary of the Invention

[0004] The first objective of this invention is to provide a new species of *Guizhou largemouth flatworm* and its DNA standard detection gene, wherein the new species of *Guizhou largemouth flatworm* has good nematode control efficacy.

[0005] The second objective of this invention is to provide the application of the above-mentioned new species of Guizhou largemouth flatworm in the biological control of predatory nematodes, especially agricultural pest nematodes.

[0006] To address the aforementioned technical problems, this invention provides a new species of *Macrostomum guizhouense* Zhang, sp. nov., whose genital spines are "J"-shaped with three bends, B2 in the opposite direction to B1, and visible scales between B2 and B3, with a pointed opening at the distal end.

[0007] This invention also provides a DNA standard detection gene for the new species of *Guizhou large-mouthed flatworm* as described above, the DNA standard detection gene for the new species of *Guizhou large-mouthed flatworm* comprising:

[0008] Its 18S rDNA sequence is shown in SEQ ID No. 1-3;

[0009] Its 28S rDNA sequence is shown in SEQ ID No. 4-6.

[0010] This invention also provides PCR primers for the standard detection gene of the new species of *Guizhou largemouth flatworm* as described above, the PCR primer sequence comprising:

[0011] 18S rDNA forward primer 200F: 5'-GGCGCATTTATTAGATCAAAACCA-3';

[0012] 18S rDNA reverse primer 1640R: 5'-GCAAGCCCCGATCCCTGTC-3';

[0013] 28S rDNA forward primer ZX-1: 5'-ACCCGCTGAATTTAAGCATAT-3';

[0014] 28S rDNA forward primer 1500R: 5'-GCTATCCTGAGGGAAACTTCG-3'.

[0015] This invention also provides an application of the new species of Guizhou largemouth flatworm as described above, or a new species of Guizhou largemouth flatworm identified by detection based on the DNA standard detection gene, in the field of biological control of nematodes.

[0016] Specifically, the nematodes include those of the genera *Rhabditis* sp. or *Meloidogynes* sp.

[0017] This invention also provides a nematode biological control agent, comprising the new species of *Platytomyces guizhouensis* or *Platytomyces guizhouensis* identified by detection based on the DNA standard detection gene.

[0018] Specifically, the new species of large-mouthed flatworm includes the new species of Guizhou large-mouthed flatworm or its eggs.

[0019] Specifically, the nematode biological control agent also includes other acceptable carriers and / or excipients compatible with the new species of *Platytomegaloides*.

[0020] Specifically, the nematode biological control agent also includes other active ingredients that have nematode control effects when combined with the new species of *Platytomyces macrocheilus*.

[0021] Specifically, the nematode biological control agent is applied to the plant rhizosphere or water bodies.

[0022] The present invention also provides a method for biological control of agricultural nematode diseases, including the step of using the nematode biological control agent.

[0023] This invention isolates Guizhou largemouth flatworm from the freshwater environment of Guizhou Province, China. This flatworm can actively prey on small rod-shaped nematodes (Rhabditis sp.) in less than 1 minute, and can be used for the biological control of nematodes in agricultural planting.

[0024] The Guizhou large-mouthed flatworm described in this invention has advantages such as high predation efficiency, strong reproductive capacity, and ease of laboratory culture. It can be used for the biological control of nematode pests in agriculture, horticulture, and aquaculture, reducing the use of chemical pesticides and has good application prospects.

[0025] This invention combines traditional morphological identification with molecular developmental analysis to identify the Guizhou large-mouthed flatworm, ensuring the accuracy of species identification. Attached Figure Description

[0026] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0027] Figure 1 The image shows the overall morphology and penile spine structure of *Guizhou flatworm*. A: Natural morphology (scale bar 200 µm); B: Enlarged head region (scale bar 50 μm); C: Tail reproductive structure (scale bar 100 µm); DF: Penile copulatory spines (scale bar 50 μm).

[0028] Figure 2 Anatomical diagrams of the reproductive system of *Guizhou flatworm*; A: Ventral view of a whole specimen, stained with hematoxylin and eosin (scale bar 200 µm); B: Magnified head region (scale bar 25 µm); C: Horizontal section showing the worm body (scale bar 100 µm); D: Tail reproductive structures (scale bar 50 µm); E: Penile spines (scale bar 25 µm).

[0029] Figure 3 Phylogenetic tree of the family Megatomidae;

[0030] Figure 4 This diagram illustrates the egg development process of the Guizhou flatworm; where A: newly laid egg (scale bar 100 µm); B: egg after 2 days of incubation (scale bar 100 µm); C: larva hatching (scale bar 50 µm); D: larva fully hatched (scale bar 50 µm).

[0031] Figure 5 The diagram shows the predatory behavior of the Guizhou large-mouthed flatworm preying on nematodes; the scale bar for A is 100 μm; and the scale bar for BD is 200 µm. Detailed Implementation

[0032] Example 1

[0033] This embodiment describes the isolation and culture of *Platycetes guinanensis*.

[0034] Aquatic plants were collected from the freshwater environment of Pingba District, Anshun City, Guizhou Province. After being filtered through a 75 μm biological sampling net and allowed to settle, Guizhou large-mouth flatworms were isolated under a stereomicroscope.

[0035] The worms were placed in 90 mm petri dishes, and their original habitat water was added. They were cultured at 25±1℃ and fed with laboratory-cultured Rhabditis sp. twice a week, with the culture water changed after each feeding. The flatworms could survive for more than a year and continue to reproduce.

[0036] In this embodiment, a species of macrotomum was isolated from the freshwater environment of Guizhou Province, China. It was identified as *Macrostomum guizhouense*, named *Macrostomum guizhouense* Zhang, sp. nov. Observation and identification revealed the following morphological characteristics: mature individuals are 1035±227 μm in length and 304±111 μm in width, with a transparent, tongue-shaped body; the body surface is covered with short cilia, and there is a pair of kidney-shaped eyes at the anterior end; the pharynx is oblong, and the mouth is located behind the eyes; the tail has mucous glands.

[0037] Example 2

[0038] This embodiment observes and identifies the reproductive system characteristics of the aforementioned macrostomum guizhouense Zhang, sp. nov.

[0039] Following the known method for preparing whole-mount specimens (Yang et al. 2020), the specific procedures were as follows: Flatworms were starved for 3 days, cleaned, placed on a glass slide, and blotted dry with filter paper. The specimens were anesthetized with 5% ethanol, flattened with a coverslip, and then transferred to a concave slide. They were then treated in fixative for 1 hour. Based on the tissue characteristics of the specimens in this study, the fixative formulation was adjusted (16 mL formaldehyde, 60 mL saturated picric acid, 6 mL glacial acetic acid, and 18 mL pure water). The specimens were washed alternately with pure water and 75% ethanol. The whole-mount slides were stained using the HE staining method.

[0040] Tissue section samples were cleaned and anesthetized as with pre-mounted slides. Fixation with fixative for 1 h was followed by routine gradient ethanol dehydration (using eosin-labeled eosin), xylene clearing, and paraffin infiltration for 1 h (57°C), resulting in sections 5 µm thick. Serial longitudinal, transverse, and horizontal sections were then performed. The tissue sections were analyzed using a one-step three-color method (Rieger et al. 1994).

[0041] Dissection of the penile canaliculi was performed using polyvinyl lactophenol (volume ratio of lactic acid:phenol = 1:1). The penile canaliculi were examined under a stereomicroscope with a cold-light digital color camera (Olympus DP72, PA, USA). Images of the live specimens, sections, and penile canaliculi were taken using a differential interference microscope (Olympus BX51, PA, USA), and features were measured using DP 2-BSW professional software (Olympus, version 2.2, PA, USA).

[0042] In this embodiment, the overall morphology and penile spine structure diagrams identified based on the squash specimens of living individuals of *Macrostomum guizhouense* are attached. Figure 1 As shown.

[0043] In this embodiment, the anatomical diagram of the reproductive system based on the identification of whole slides and sections of Macrostomum guizhouense is attached. Figure 2 As shown.

[0044] As can be seen, the Guizhou flatworm of this invention is hermaphroditic, with a "J"-shaped phallus that has three bends (B1, B2, B3). B2 is in the opposite direction to B1, and B3 bends inward at about 60°. Scales are visible between B2 and B3. The distal end is pointed, and the inner side is arrow-shaped. This phallus morphology is different from other known flatworm species.

[0045] Example 3

[0046] This embodiment uses standard DNA testing to identify the aforementioned macrostomum guizhouense Zhang, sp. nov.

[0047] The molecular characteristics of the Guizhou flatworm were determined as follows:

[0048] Its 18S rDNA sequence is shown in SEQ ID No. 1-3;

[0049] Its 28S rDNA sequence is shown in SEQ ID No. 4-6.

[0050] SEQ ID No. 1 (M18S-1):

[0051]

[0052] SEQ ID No.2(M18S-2):

[0053]

[0054] SEQ ID No.3(M18S-3):

[0055]

[0056] SEQ ID No.4(M28S-1):

[0057]

[0058] SEQ ID No.5(M28S-2):

[0059]

[0060] SEQ ID No.6(M28S-3):

[0061]

[0062] Phylogenetic analysis showed that this new species is most closely related to Macrostomum zhaoqingense and Macrostomum sinenese, but clusters independently into a single lineage.

[0063] Example 4

[0064] This embodiment is based on the DNA standard detection of the aforementioned Flatworm in Embodiment 2 to identify the large-mouthed flatworm.

[0065] The identification method described in this embodiment involves designing primers to amplify 18S rDNA and 28S rDNA, then using NCBIBLAST to obtain homologous sequences, and constructing a phylogenetic tree based on the selected sequences to identify new species.

[0066] The method for obtaining 18S rDNA and 28S rDNA sequences in this embodiment is as follows.

[0067] DNA extraction, amplification, and sequencing were performed according to the method of Yang et al. (Yang et al. 2020). Specimens were starved in clean habitat water for 3 days. Three specimens were taken from the starved specimens, treated with liquid nitrogen, and sequenced using EZNA. TM DNA was extracted using MolluscDNA KIT (omega, Norcross, GA, USA). 18S rDNA primers and 28S rDNA primers, along with the PCR procedure, are shown in Table 1 below.

[0068] Table 1. PCR primers and PCR procedure

[0069]

[0070] All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. Add Premix Taq TM (TakaRa, Dalian, China) PCR was performed, and the obtained PCR products were ligated and cloned into pMD. TM The 19-T Vector Cloning kit (TakaRa, Dalian, China) was used to transform the bacteria into competent *Escherichia coli* Top 10. The resulting bacterial culture, after plating, single-clone picking, and culturing, was sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0071] In this embodiment, the 18S rDNA and 28S rDNA sequences used as identification criteria for the detection gene have been uploaded to the GeneBank database. The sequences used for molecular systematic analysis are all from GeneBank. Homologous sequences were obtained using NCBI BLAST, and the results are shown in Table 2 below.

[0072] Table 2. GenBank accession numbers of sequences involved in phylogenetic analysis.

[0073]

[0074] Furthermore, phylogenetic analysis was conducted based on the aforementioned identification methods.

[0075] This study constructed a phylogenetic tree using the 18S rDNA and 28S rDNA gene sequences of 24 species of Macrostomidae, 3 species of Dolichomacrostomum, and 1 species of Microstomum, as shown in the attached figure. Figure 3 As shown. Among them, *Haplopharynx rostratus* was selected as an outgroup. After using ClipKIT to cleave the sequences, the final lengths of the 18S rDNA and 28S rDNA sequences were 1649 bp and 1064 bp, respectively. According to the Modelfinder selection results (Kalyaanamoorthy et al. 2017), the optimal models for 18S and 28S rDNA were GTR+F+I+G4 and GTR+F+R6, respectively, suitable for Bayesian inference and maximum likelihood analysis. *Macrostomum guizhouense* is located in *Macrostomum* and is most closely related to *Macrostomum zhaoqingense* and *Macrostomum sinenese*.

[0076] The phylogenetic tree was generated by splicing 18S and 28S rDNA. The topological structures of both Bayesian inference and maximum likelihood methods were identical, so the results of the two methods were combined. As the phylogenetic tree shows, the species of the Macrostomidae family are divided into three main branches. Outgroups are located at the base of the phylogenetic tree, forming independent branches. In the first branch (Macrostomum), three individuals of *Macrostomum guizhouense* cluster together (posterior probability = 0.92, unfolding value = 76%), and it clusters with *Macrostomum zhaoqingense* as a sister group with extremely high support (posterior probability = 1.00, unfolding value = 100%). Furthermore, this lineage also forms a sister group with *Macrostomum sinenese* and *Macrostomum chongqingense*, with high support (posterior probability = 1.00, unfolding value = 90%). The phylogenetic tree results support the status of *Macrostomum guizhouene* as a new species of Macrostomidae.

[0077] Therefore, the DNA standard detection gene described in this invention supports the new species status of *Guizhou large-mouthed flatworm*.

[0078] Example 5

[0079] This embodiment is based on the aforementioned Macrostomum guizhouense Zhang, sp. nov., and its reproduction and hatching observation.

[0080] Sexually mature *Guizhou flatworm* were cultured individually on concave glass slides, and their oviposition behavior was observed. The results are attached. Figure 4 As shown.

[0081] As can be seen, the eggs of the large-mouthed flatworm are pale yellow, nearly spherical, and hatch in about 3 days. On the first day, the eggs are filled with irregular contents; on the second day, a distinct embryo appears; and on the third day, the larva hatches, has eyespots, and can move freely.

[0082] The Guizhou flatworm, under laboratory conditions, feeds on nematodes and can survive and reproduce normally for a long time. The egg hatching cycle is 3±1 days, and each egg hatches into one larva.

[0083] Example 6

[0084] This embodiment observes the predatory behavior of the aforementioned macrotomum guizhouense Zhang, sp. nov., and the results are attached. Figure 5 As shown.

[0085] It is evident that a flatworm that has been starving for a day can rapidly prey on nematodes freshly placed in a petri dish. During feeding, the flatworm senses its prey through rod-shaped receptors. Upon approaching the nematode, its longitudinal slit-like opening continuously expands and contracts to suck in the nematode. Through the continuous expansion and contraction of its pharynx, it engulfs the nematode and swallows it into its intestine. Microscopic observation revealed the morphology of the nematode entering the intestine; even after being swallowed, the nematode continues to struggle by constantly wriggling. At room temperature (24 ℃), it was observed that the time from the flatworm's initial contact with the nematode to its complete ingestion into the intestine was less than 1 minute (n=5). Microscopic observation showed that the flatworm could continuously prey on 3-4 nematodes. Several minutes later, the nematodes in the flatworm's intestine were digested and decomposed.

[0086] In summary, this flatworm is capable of actively preying on nematodes of the genus *Rhabditis*. The predation process includes: sensing the prey, expanding the mouth to grasp the nematode, and contracting the pharynx to swallow it into the intestine. The nematode is rapidly digested in the intestine, and the entire process takes no more than one minute.

[0087] Example 7

[0088] This embodiment prepares a biocontrol agent based on *Guizhou largemouth flatworm* or its eggs as an active ingredient or as the sole active ingredient. The biocontrol agent comprises an effective amount of *Guizhou largemouth flatworm* or its eggs. *Guizhou largemouth flatworm* or its eggs can prey on nematodes, especially *Rhabditis* sp. or *Meloidogyne* sp. nematodes.

[0089] In this embodiment, the biocontrol agent may be selected as the Guizhou largemouth flatworm or its eggs as the sole active ingredient; it may also include other active ingredients known in the art for nematode control.

[0090] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A new species of Guizhou flatworm, characterized in that, It is named Macrostomum guizhouense Zhang,sp. nov. Its penile spines are "J" shaped with three bends, B2 and B1 are in opposite directions, there are visible scales between B2 and B3, and the distal opening is pointed.

2. A DNA standard detection gene for a new species of *Guizhou flatworm* as described in claim 1, characterized in that, The DNA standard detection genes of the new species of *Guizhou largemouth flatworm* include: Its 18S rDNA sequence is shown in SEQ ID No. 1-3; Its 28S rDNA sequence is shown in SEQ ID No. 4-6.

3. A PCR primer for the standard detection gene of the DNA of a new species of *Platycetes guishanensis* as described in claim 2, characterized in that, The PCR primer sequences include: 18S rDNA forward primer 200F: 5'-GGCGCATTTATTAGATCAAAACCA-3'; 18S rDNA reverse primer 1640R: 5'-GCAAGCCCCGATCCCTGTC-3'; 28S rDNA forward primer ZX-1: 5'-ACCCGCTGAATTTAAGCATAT-3'; 28S rDNA forward primer 1500R: 5'-GCTATCCTGAGGGAAACTTCG-3'.

4. The application of a new species of Guizhou largemouth flatworm as described in claim 1, or a new species of Guizhou largemouth flatworm identified by detection based on the DNA standard detection gene as described in claim 2, in the field of biological control of nematodes.

5. The application according to claim 4, characterized in that, The nematodes include those of the genera *Rhabditis* sp. or *Meloidogyne* sp.

6. A biological control agent for nematodes, characterized in that, The species includes the new species of *Platyceps guishanensis* as described in claim 1, or *Platyceps guishanensis* identified by detection based on the DNA standard detection gene described in claim 2.

7. The nematode biological control agent according to claim 6, characterized in that, The new species of *Platyceps militaris* includes the new species of *Platyceps militaris* from Guizhou or its eggs.

8. The nematode biological control agent according to claim 6 or 7, characterized in that, The nematode biocontrol agent also includes other acceptable carriers, excipients, or other active ingredients that have a nematode-controlling effect and are compatible with the new species of *Platytomyces macrocheilus*.

9. The nematode biological control agent according to claim 6 or 7, characterized in that, The nematode biological control agent is applied to the plant rhizosphere or water bodies.

10. A method for biological control of agricultural nematodes, characterized in that, The step includes using the nematode biological control agent according to any one of claims 5-9.

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