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Genetical and Morphological Identification of Prosthogonimus pellucidus (Digenea, Prosthogonimidae) in Grus japonensis
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Article

Genetical and Morphological Identification of Prosthogonimus pellucidus (Digenea, Prosthogonimidae) in Grus japonensis

by
Yu Cao
1,2,*,†,
Ye Li
1,†,
Zhong-Yan Gao
3,
Xian-Guang Zhang
3,
Bo-Tao Jiang
1,2 and
Hong-Bao Wang
1
1
Heilongjiang Academy of Agricultural Sciences Branch of Animal Husbandry and Veterinary Branch, Qiqihar 161005, China
2
Heilongjiang Provincial Key Laboratory of Veterinary Drugs, Qiqihar 163313, China
3
Heilongjiang Zhalong National Natural Reserve Administration, Qiqihar 161000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biology 2024, 13(11), 900; https://doi.org/10.3390/biology13110900
Submission received: 19 September 2024 / Revised: 27 October 2024 / Accepted: 28 October 2024 / Published: 5 November 2024
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Simple Summary

Species of the family Prosthogonimidae are considered the most pathogenic trematodes of poultry and wild birds worldwide, causing heavy economic losses in many countries. The authors of this study conducted morphological and molecular identification of Prosthogonimus pellucidus. These data provide significant molecular markers for the study of taxonomy, population genetics, and systematics of Prosthogonimidae.

Abstract

Species of the family Prosthogonimidae are considered the most pathogenic trematodes of poultry and wild birds worldwide, causing heavy economic losses in many countries. Prosthogonimosis was a common parasitic disease of Grus japonensis (Müller, 1776) which caused inflammation of the cloaca and bursa of Fabricius and even death. Morphological identifications of Prosthogonimus species are easily confusing; therefore, molecular characterization is used for discrimination. The present study was conducted to identify Prosthogonimus species at Zhalong National Nature Reserve, northeast of China. Considering the morphological variability and wide host range of individual Prosthogonimus species, a combination of both morphological and molecular analyses is indispensable for the valid identification of this parasite and the internal transcribed spacer (ITS) region was amplified for the sequence analysis and phylogenetic analysis. The results of molecular analysis together with phylogenetic reconstruction indicated that the Prosthogonimus pellucidus (von Linstow, 1873) in this study form a single cluster with P. pellucidus, revealing potentially high diversity within the genus Prosthogonimus. Classification of Prosthogonimus species seems to be unrelated to the host and may be related to geographical location. These data provide a significant resource of molecular markers for studying the taxonomy, population genetics, and systematics of Prosthogonimidae.

1. Introduction

Species of the family Prosthogonimidae are considered the most pathogenic poultry trematodes worldwide, affecting particularly free-range poultry and wild birds. They have been reported in many regions worldwide, including Asia, Europe, Africa, and America [1,2,3,4,5]. The Prosthogonimus species that have been reported in China include Prosthogonimus ovatus (Rudolphi, 1803), Prosthogonimus cuneatus (Rudolphi, 1809) and Prosthogonimus pellucidus (von Linstow, 1873) [1,6]. P. pelucidus is a common Prosthogonimus species and is distributed in the south, east, northeast, and western regions of China. The life cycle of Prosthogonimus spp. is shown in Figure 1. Qiu and Liu (1983) identified Gabbia fuchsiana (Möllendorf, 1888) as the first intermediate host of P. pellucidus in China [6]. The snail takes up the trematode eggs deposited in the water and the miracidium that hatched in the intestine of the snail migrates into the hepatopancreas. Here, sporocysts are produced and cercariae are released into the water. Final hosts become infected by dragonflies or damselflies (Odonata), which contain metacercariae of Prosthogonimus spp. Adults of Prosthogonimus species mainly parasitize in the cloaca, oviduct, and bursa Fabricii of poultry, and excrete eggs in the host’s feces [7]. Poultry infected with Prosthogonimus species show inflammation of the oviduct and bursa of Fabricius, and may lay eggs with soft shells or without any shell at all [5]. In addition, Prosthogonimus species may rarely also infect mammals, where they reside in the body cavity, intestine, or liver, and lead to peritonitis [8,9]. It has been reported that a nine-month-old infant was infected by Prosthogonimus sp. in Indonesia [10].
In spite of the morbidity and economic loss of Prosthogonimosis, there has been an important controversy about the taxonomy of Prosthogonimus species [8]. Most studies have mainly focused on the morphology and epidemiology of Prosthogonimus species [11,12]. In order to characterize different species in endemic areas, genetic studies of this parasite can be conducted, as genetic methods provide more reliable results. Generally, ITS rDNA sequences show less intra-specific variation than inter-specific variation; therefore, they are considered reliable markers for species differentiation. Some molecular-based studies of internal transcribed spacers could provide valuable genetic markers for species identification of trematodes [13,14,15]. The present research aimed to study ITS2 rDNA sequences as a genetic marker for the genetic characterization of Prosthogonimus species, the reconstruction of its phylogenetic relationship, and a comparison of this characterization with previously reported results from P. pelucidus obtained from G. japonicas and other avian species.

2. Materials and Methods

2.1. Worms Collection

Adult worms were all collected from the cloaca, oviduct, and bursa Fabricii of naturally infected G. japonensis in the Zhalong National Nature Reserve, which followed the wildlife protection law of the People’s Republic of China (a draft of an animal protection law in China released on 2018). A total of 18 trematodes were collected in this study. The worms were washed with normal saline solution and preserved in 70% alcohol at −20 °C.

2.2. Morphological Identification

One adult fluke was prepared for morphological studies. For this, the fluke was placed between two glass slides and placed in 70% ethanol for one month. During this month, the force on the slide was gradually increased. The worms were in 50% ethanol and 30% ethanol for 1 h, respectively, then soaked in purified water for 2 h, and stained with hematoxylin solution overnight. The staining solution was poured and purified water, which was used to remove the floating color, and then the worms were put into 30%, 50%, and 70% gradient ethanol, successively. The soaked time of each grade of ethanol was 40 min, 35 min, and 55 min, respectively. Acid alcohol was used for decolorization for 30 s, followed by 80%, 95%, and 100% gradient ethanol. The soaked time of each grade of ethanol was 40 min, 30 min, and 60 min, respectively. The trematode was cleared with xylene for 16 h, embedded in Canada balsam, and examined under a light microscope.

2.3. Genomic DNA Extraction and PCR Amplification

Total genomic DNA was extracted from 5 adult worms randomly selected from 18 according to the instructions for the TIANamp Genomic DNA Kit (Tiangen, Beijing, China). The nucleic acid concentration of the extracted DNA was detected, and then the DNA was stored at −20 °C for genetic analysis. The PCR method was used to amplify the ITS sequence of the Prosthogonimus species. The PCR reaction was based on previous study primers: NC5: 5′-GTAGGTGAACCTGCGGAACGATCATT-3′, NC2: 5′-TTAGTTTCTTTTCCTCCGCT-3′ [16]. The 25 µL PCR reactions were performed using 18.3 µL of distilled water, 2 µL of dNTP Mixture (2.5 mM), 2.5 µL of 10 × Ex Taq buffer, 0.5 µL of each primer (25 mM), 1 µL of extracted DNA, and 0.2 µL of Ex Taq DNA polymerase (5 U/µL). The DNA template of positive control used fluke DNA stored in the laboratory, previously. The size of the positive control was 1269 bp. The DNA template of negative control used distilled water, under the following conditions: 94 °C for 5 min (initial denaturation), then 94 °C for 30 s (denaturation), 50–65 °C for 1 min (annealing), and 72 °C for 1 min 30 s (extension) for 35 cycles, and a final extension at 72 °C for 10 min.

2.4. Sequence Alignments and Phylogenetic Analyses

The positive bands of PCR products amplified by NC2 and NC5 primers were good, which were sequenced using the Sanger method. Sequences were assembled manually and aligned against the fluck of Microphalloidea in GenBank, to identify gene boundaries, using the program DNAStar v. 5.0 [17]. The edited sequence was submitted in GenBank for the accession ID number. The intra-specific variations and inter-specific variation were calculated using MEGA v. 5.0 and MegAlign v. 5.01 [17,18]. Comparisons were made based on nucleotide sequence difference, determined from the ITS2 sequence among P. cuneatus (OQ344776.1), P. ovatus (KP192735.1), P. pellucidus (KP192732.1), and Prosthogonimus rarus (Braun, 1901) (KP192728.1). The AT and GC content of the ITS sequences were calculated using DNAStar v. 5.0 [17]. The forward, reverse, complement, and palindromic repeats of the ITS sequences of P. pellucidus were examined by REPuter [19]. These repeats were ≥10 bp with a maximum computed repeats of 100 bp.
In addition to our P. pellucidus data, the 17-member phylogenetic dataset contains the following: P. ovatus (KP192735) from Passer domesticus (Poland); P. ovatus (KP192733) from P. domesticus (Poland); P. ovatus (KP192727) from Anas platyrhynchos (Czech Republic); P. ovatus (KP192723) from Anas strepera (Czech Republic); P. ovatus (KP192722) from Aythya ferina (Czech Republic); P. ovatus (KP192730) from A. platyrhynchos (Czech Republic); P. ovatus (KP192731) from A. platyrhynchos (Czech Republic); P. rarus (KP192728) from A. platyrhynchos (Czech Republic); P. rarus (KP192726) from A. platyrhynchos (Czech Republic); P. rarus (KP192724) from Anas clypeata (Czech Republic); P. cuneatus (KP192738) from Turdus merula (Czech Republic); P. cuneatus (KP192736) from T. merula (Czech Republic); P. cuneatus (KP192729) from A. platyrhynchos (Czech Republic); P. cuneatus (KP192725) from A. platyrhynchos (Czech Republic); Prosthogonimus falconis (OK044379) from Falco peregrinus (The United Arab Emirates); P. pellucidus (KP192734) from P. domesticus (Poland); P. pellucidus (KP192732) from A. platyrhynchos (Czech Republic). And Collyriclum faba (JQ231122) from Saxicola rubetra was included as an outgroup. Phylogenetic trees were all reconstructed using maximum parsimony (MP) methods. MP methods were performed using the Fitch criterion within PAUP v. 4.0 Beta 10 [20], and bootstrap support values were calculated in PAUP from 1000 bootstrap replicates with 10 random additions per replicate. Phylograms were viewed and drawn using FigTree V. 1.42 [21].

3. Results and Discussion

3.1. The Morphological Features of P. pellucidus

The examined trematode is pear-shaped, 4.31 mm long with a rounded posterior and a pointed anterior end (Figure 2). The maximum width was 2.5 mm. Minute tegumental spines were observed at the anterior part. The oval ventral sucker (0.61 × 0.64 mm) is situated in the first third of the body. The subterminal oral sucker measures 0.42 × 0.48 mm and is followed by a small spherical pharynx. The two intestinal branches extend nearly to the posterior end. The excretory vesicle is Y-shaped with long branches. The two oval, unlobed testes are situated in a parallel position posterior to the acetabulum in mid-body position and measure 0.58 × 0.35 mm and 0.55 × 0.29 mm. The genital pore is situated lateral to the oral sucker on the anterior end. The deeply lobed ovary is at the posterior edge of the acetabulum. The follicular extra-caecal yolk glands commence from the posterior margin of the acetabulum and extend to the border between second and third portion of the body. The heavily coiled uterus fills the posterior body posterior to acetabulum. A single uterus lobe extends dorsal to the ventral sucker to the genital pore. Operculated eggs in the distal uterus are 27 × 12 µm and contain a fully developed miracidium. According to the above morphological characteristics and the judgment criteria in reference [8,11], it was preliminarily identified as P. pellucidus. We attempted to elucidate the morphological characteristics of P. pellucidus that might increase our knowledge and understanding of its morphology. Morphological characteristics are generally used to identify adult trematode. However, sometimes morphological parameters are not sufficient as a basis for species differentiation. Thus, molecular approaches are considered for working on the evolution and systematics of trematodes.

3.2. The ITS Sequence Features of P. pellucidus

The positive bands of PCR products amplified were good, with a size of approximately 1200 bp, which were sequenced using the Sanger method (Figure S1). Five sequences of the ITS rRNA gene were examined in this study, which were submitted to GenBank. The accession numbers were PP955191, PP958838, PP956936, PP956935, and PP956934, respectively. The lengths of the five partial ITS rDNA sequences obtained were 1183 bp, 1209 bp, 1269 bp, 1261 bp, and 1265 bp, respectively. The ITS sequences contain three genes: internal transcribed spacer-1 (ITS1), 5.8S rDNA-ITS sequence (5.8S), and internal transcribed spacer-2 (ITS2). The lengths of the five partial ITS1 sequences in this study were 852 bp, 854 bp, 900 bp, 901 bp, and 935 bp, respectively. The lengths of the five complete 5.8S sequences in this study were all 107 bp. The lengths of the five partial or complete ITS2 sequences in this study were 224 bp, 227 bp, 248 bp, 254 bp, and 257 bp, respectively.
For 5.8S and ITS2 sequences, the intra-specific variations within P. pellucidus in this study were 0%. And the intra-specific variations within P. pellucidus in this study of ITS1 sequences was 0–7%. P. pellucidus of this study was genetically characterized through ITS rDNA sequences at Zhalong National Nature Reserve, China. Based on the specific location of the study and its only involving one site and one species of avian host, this study had limitations. No separate ITS1 rDNA sequence of P. pellucidus was available in the NCBI database. Therefore, the ITS2 rDNA sequence was used for the analysis of Prosthogonimidae sequence differences. Among the five Prosthogonimidae species, the sequence differences in the ITS2 sequence were 2.2−16.0% at the nucleotide level. The P. ovatus sequences were the most different from P. pellucidus in this study, and these differences were 12.9%. The P. pellucidus sequences were the least different from P. pellucidus in this study, and these differences were 2.2%.
The five ITS sequences of this study had nucleotide compositions that were biased toward C and G, with an overall C+G content of 50.62–51.94% and A+T content of 48.06–49.38%. The ITS1 sequence nucleotide compositions were biased toward both C and G, with an overall C+G content of 50.82–52.44%, and A+T content of 47.56–49.18%. The 5.8S sequence nucleotide compositions were both biased toward C and G, with an overall C+G content of 51.4% and A+T content of 48.6%. The ITS2 sequence nucleotide composition was biased toward A+T, with an overall A+T content of 49.61–50.89% and C+G content of 49.11–50.39%.
In the current study, forty-two repeat sequences were found in total, and the forward, reverse, complement, and palindromic repeats of the ITS sequences were thirty-four, two, two, and four, respectively. The repeat sequences of ITS1 were the most, with thirty-nine repeat sequences (Table 1). Internal repeats appear to be characteristic of the ITS1 evolution in different groups of organisms [22].

3.3. Phylogenetic Analyses

Phylogenetic analyses of nucleotide sequences from 23 trematode ITS2 sequences were performed using the MP approach (Figure 3). This phylogenetic MP tree splits into two large clades. One clade contains P. ovatus and P. rarus, and the other clade contains P. cuneatus, P. falconis., and P. pellucidus. In the first clade, different hosts of P. ovatus (Poland and Czech Republic) cluster together, and different hosts of P. rarus (Czech Republic) cluster together, respectively. In the second clade, different hosts of P. cuneatus (Czech Republic) cluster together. P. pellucidus from G. japonensis (China) in this study cluster together, and form a sister taxa with P. pellucidus from P. domesticus and A. platyrhynchos (Poland and Czech Republic). Phylogenetic analyses revealed that classification of Prosthogonimus species seems to be unrelated to the host and may be related to geographical location.
The Prosthogonimus pellucidus of this study was most closely related to P. pellucidus in the previous study, and then to P. cuneatus, P. ovatus, and P. rarus. This is similar to a previous study using concatenated amino acid sequence data representing 12 protein-coding genes, in which P. cuneatus and P. pellucidus clustered together [23]. This is consistent with a previous study using ITS2, CO1, and ND1 sequences in which P. pellucidus and P. cuneatus cluster together on one branch, and P. rarus and P. ovatus cluster together on one branch [24]. In the first clade, P. ovatus and P. rarus cluster together. It is also consistent with a previous study using ITS2, CO1, and ND1 sequences, in which the phylogenetic relationships of four Prosthogonimus spp. were reconstructed and P. ovatus and P. rarus formed one clade [8]. This is also consistent with a previous study using 28S and CO1 sequences, wherein P. cuneatus and cercaria of P. pellucidus clustered together [25]. Further characterization of Prosthogonimidae phylogeny will need to wait until additional genomic trematode data have been deposited in GenBank.

4. Conclusions

The present study determined the partial sequence of five ITS rDNA sequences of P. pellucidus and conducted sequence analysis and phylogenetic analysis. Possibly, the ITS1 marker is a useful genetic marker to study genetic variation with 5.8S and ITS-2 markers, because the ITS1 sequence has repeats and mutation sites, but there are few ITS1 sequences of Prosthogonimidae in the NCBI database; this study added the ITS1 sequence to the database for P. pellucidus. Phylogenetic analyses indicate that classification of Prosthogonimus species seems to be unrelated to the host and may be related to geographical location, and P. pellucidus is the dominant species in this region. From ecological or phylogenetical viewpoints, more detailed genetic analyses will give valuable information, and these data will provide a significant resource regarding molecular markers for studying the taxonomy, population genetics, and systematics of Prosthogonimidae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology13110900/s1, Figure S1: Agarose gel electropheoresis of ITS PCR products of P. pellucidus samples M: maker; 1–5: PCR products of P. pellucidus ITS rDNA sequence. 6: positive control; 7: negative control.

Author Contributions

All authors contributed to this study’s conception and design. Y.C. and Y.L. developed the rationale of the study and wrote the manuscript. Y.C. designed and performed most of the experiments with contributions from Y.L., Z.-Y.G. and X.-G.Z.; B.-T.J. and H.-B.W. supervised the study. All authors have read and agreed to the published version of the manuscript.

Funding

Open access funding provided by the Heilongjiang Academy of Agricultural Sciences. This work was supported by the Heilongjiang Province agricultural science and technology innovation leapfrog project agricultural science and technology basic innovation project (Excellent Young Scholars) (Project number: CX23BS06).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no competing interests.

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Biology 13 00900 g001
Figure 1. The life cycle of Prosthogonimus spp.
Figure 1. The life cycle of Prosthogonimus spp.
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Figure 2. Prosthogonimus pellucidus. Scale bar: 0.5 mm.
Figure 2. Prosthogonimus pellucidus. Scale bar: 0.5 mm.
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Figure 3. Genetic relationships of P. pellucidus with other representative trematodes based on ITS2 sequence data. Phylogenetic analyses used maximum parsimony (MP), with C. faba as outgroup. Scale bar indicates posterior probability. The red font represents the trematode in this study.
Figure 3. Genetic relationships of P. pellucidus with other representative trematodes based on ITS2 sequence data. Phylogenetic analyses used maximum parsimony (MP), with C. faba as outgroup. Scale bar indicates posterior probability. The red font represents the trematode in this study.
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Table 1. Information of repeat sequences in the ITS rDNA of Prosthogonimus pellucidus.
Table 1. Information of repeat sequences in the ITS rDNA of Prosthogonimus pellucidus.
IDRepeat
Start 1		TypeSize
(bp)		Repeat
Start 2		Repeat
Distance		Gene
1603Forward106680ITS1
21066Forward1011790ITS2
3167Forward262610ITS1
4167Forward263100ITS1
5167Forward263590ITS1
6167Forward264080ITS1
7167Forward264570ITS1
872Forward303060ITS1
972Forward303550ITS1
1072Forward304040ITS1
1172Forward304530ITS1
12119Forward303060ITS1
13119Forward303550ITS1
14119Forward304040ITS1
15119Forward304530ITS1
16123Forward301670ITS1
17210Forward303060ITS1
18210Forward303550ITS1
19210Forward304040ITS1
20210Forward304530ITS1
21257Forward304530ITS1
22257Forward353060ITS1
23257Forward353550ITS1
24257Forward354040ITS1
25347Forward384450ITS1
26396Forward384450ITS1
2761Forward412460ITS1
28293Forward434400ITS1
2961Forward451080ITS1
30298Forward483960ITS1
31167Forward732140ITS1
3276Forward771670ITS1
3361Forward881990ITS1
34298Forward973470ITS1
35793Reverse1011600ITS1; ITS2
36638Reverse176380ITS1
37656Complement108650ITS1
38576Complement1111510ITS1; ITS2
39637Palindromic1010480ITS1; ITS2
401084Palindromic101084 0ITS2
411147Palindromic1011470ITS2
42489Palindromic124890ITS1
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MDPI and ACS Style

Cao, Y.; Li, Y.; Gao, Z.-Y.; Zhang, X.-G.; Jiang, B.-T.; Wang, H.-B. Genetical and Morphological Identification of Prosthogonimus pellucidus (Digenea, Prosthogonimidae) in Grus japonensis. Biology 2024, 13, 900. https://doi.org/10.3390/biology13110900

AMA Style

Cao Y, Li Y, Gao Z-Y, Zhang X-G, Jiang B-T, Wang H-B. Genetical and Morphological Identification of Prosthogonimus pellucidus (Digenea, Prosthogonimidae) in Grus japonensis. Biology. 2024; 13(11):900. https://doi.org/10.3390/biology13110900

Chicago/Turabian Style

Cao, Yu, Ye Li, Zhong-Yan Gao, Xian-Guang Zhang, Bo-Tao Jiang, and Hong-Bao Wang. 2024. "Genetical and Morphological Identification of Prosthogonimus pellucidus (Digenea, Prosthogonimidae) in Grus japonensis" Biology 13, no. 11: 900. https://doi.org/10.3390/biology13110900

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