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Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 23 of 38
Volume 4, Issue 4, October 2022
Received : 26 March 2022
Accepted : 22 August 2022
Published : 05 October 2022
doi: 10.33472/AFJBS.4.4.2022.23-38
Article Info
© 2022 Martin L. Nelwan. This is an open access article under the
CC BY license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Abstract
Introduction: In this study, I report the progress of Schistosomiasis japonica that
focused on dispersion of Schistosoma japonicum, genus Oncomelania, and use of
genetic manipulations on Oncomelania lorelindoensis for eliminating schistosomiasis
japonica in Central Sulawesi as objectives of this study. Results: Results of
Nucleotide BLAST showed that Sulawesi’s S. japonicum originated from Japan
and China. Results also showed that Southeast Asian Oncomelania is closer to O.
hupensis robertsoni (Oncomelania robertsoni) than to O. hupensis or O. minima.
Elimination of S. japonicum parasite in O. lorelindoensis can occur in the field using
anti-Schistosoma inoculated into female ovary O. lorelindoensis with CRISPR/Cas9
system. The progeny of transgenic snails in each generation can be calculated by
using the mathematical ideas. Mathematical ideas include total F1 = 3n, F1 = ([3n –
1] : 1), total F2 is Fn+1 = 6n, and Fn+1 = ([6n – 1] : 1). Conclusion: Nucleotide BLAST
results showed that ancestors of Sulawesi’s S. japonicum originated from Japan
and China. Oncomelania lorelindoensis is the intermediate host of Sulawesi’s S.
japonicum. Any transgenic snail crossed with wild-type O. lorelindoensis can result
in S. japonicum-resistant snails.
Keywords: CRISPR/Cas9, Oncomelania, Oncomelania lorelindoensis, Schistosoma
japonicum, Schistosomiasis
1. Introduction
Blood flukes of the genus Schistosoma cause schistosomiasis infection. Genus Schistosoma that can infect humans
include several species. These include Schistosoma haematobium, S. intercalatum, Schistosoma japonicum, S.
malayensis, S. mansoni, and S. mekongi (Butrous, 2019; El Ridi et al., 2012; and Neves et al., 2015). Schistosoma
infects about 220 to 230 million people (Ferrari et al., 2021; Nelwan, 2019; Sanches et al., 2021; and Zhou et al.,
2020). Schistosomiasis causes 24,072 to 200,000 deaths annually (World Health Organization, 2021), and 779
million people are at risk. In addition, schistosomiasis causes a worldwide burden of 3.3 million disability-
adjusted life years. Schistosomiasis occurs in Africa, Asia, South America, and several Caribbean islands. It
* Corresponding author: Martin L. Nelwan, Animal Science – Other, Nelwan Institution for Human Resource Development, Palu, Central
Sulawesi, Indonesia. E-mail: mlnelwan2@gmail.com
2663-2187/© 2022. Martin L. Nelwan. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
African Journal of Biological Sciences
ISSN: 2663-2187
Journal homepage: http://www.afjbs.com
Indonesia Schistosoma japonicum: Origin, genus oncomelania, and
elimination of the parasite with cluster genes inoculated into female
Oncomelania lorelindoensis via CRISPR/Cas9 system
Martin L. Nelwan1*
1Animal Science – Other, Nelwan Institution for Human Resource Development, Palu, Central Sulawesi, Indonesia.
E-mail: mlnelwan2@gmail.com
Research Paper Open Access
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38
https://doi.org/10.33472/AFJBS.4.4.2022.23-38
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 24 of 38
can also occur in non-developing countries. Schistosomiasis can spread through water-based development
projects, immigration (Nelwan, 2019), and travelling.
Genome sequences on phylogenic showed that S. japonicum originated from China. S. japonicum originated
from the Middle and Lower (ML) reaches of the Yangtze River. From the Yangtze River, S. japonicum spread out
to Japan, the mountainous areas of China, and then to the Philippines and Indonesia (Yin et al., 2015). S.
japonicum has at least 46 species of mammalian definitive hosts (Gordon et al., 2019). Mammalian definitive
hosts can include human, mice and wild pigs. In Indonesia, endemic areas of S. japonicum are in the Bada
Valley, the Lindu Valley, and the Napu Valley of Central Sulawesi (Budiono et al., 2019; Nelwan, 2019; and
Nurwidayati et al., 2018).
Nucleotide BLAST can provide evidences for the spread of S. japonicum and the genus Oncomelania. For
example, I found that identity percentages of S. japonicum parasites in Yangtze River (i.e., KU196321.1) against
the Philippines (KU196379.1), and Indonesia (KU196348.1) were 99.61% and 99.63%. It suggested that S.
japonicum first reached Indonesia, then the Philippines.
The genus Oncomelania is the intermediate host of S. japonicum. It consists of Oncomelania hupensis, O.
hupensis lindoensis (O. lorelindoensis), O. hupensis quadrasi (O. quadrasi), and O. minima. Oncomelania hupensis
consisted of O. hupensis chiui, O. hupensis formosana, O. hupenseis hupensis, O. hupensis nosophora, O. hupensis
robertsoni (O. robertsoni), and O. hupensis tangi. The East to West hypothesis suggests that precursors of Oncomelania
originated from Australia (Attwood et al., 2015) and Borneo-Philippine Island (Liu et al., 2014) spread to Japan.
After reaching Japan, it gave rise to O. hupensis in China. Oncomelania hupensis re-colonized Japan, the Philippines
and Sulawesi to replace antecedent form (Attwood et al., 2015). There are two species of the genus Oncomelania:
Oncomelania hupensis and O. minima. Oncomelania hupensis consists of five subspecies. Oncomelania minima
(Kameda and Kato, 2011) does not have any subspecies.
Nucleotide BLAST of ML Yangtze’s O. hupensis hupensis (KR002674.1) had identity percentages of 93.97%
with Japan’s Oncomelania (KR002673.1) and 84.40% with the Philippines’s Oncomelania (DQ112287.1). In
addition, the sequence results showed that the Philippines’s Oncomelania was closer to West China’s O.
robertsoni (87.25%; KR002675.1) than to China’s O. hupensis (84.48%; GU367391.1). This suggests that the
taxonomy of the genus Oncomelania should be reconsidered.
Praziquantel is the only effective drug for treating all Schistosoma species (El-Nour and Fadladdin, 2021;
and Nelwan, 2019). It has been available in the market since 1988. Praziquantel only kills adult worms, and
cannot kill schistosomula and juvenile worms. In addition, treatment of schistosomiasis with only one drug
for more than thirty years can result in resistant to that drug (El-Nour and Fadladdin, 2021; and Tekwu et al.,
2017). It seems that it is important to find a new method for controlling schistosomiasis. To anticipate resistance
to praziquantel, genetic manipulation techniques can be used. Genetic manipulation techniques can include
such as the adeno-associated virus (AAV) and clustered regularly interspaced short palindromic repeats
(CRISPR/Cas9) system (Nelwan, 2021a). The use of CRISPR/Cas9 system and AAV vectors has become
common in genetic manipulations (Nelwan, 2020; and Nelwan, 2021b). The CRISPR/Cas9 can deactivate the
gene for omega-1 ribonuclease S. mansoni and create parasites not produce omega-1 ribonuclease, or very little
of it (Ittiprasert et al., 2019; and McVeigh and Maule, 2019). It suggests that CRISPR/Cas9 can also deactivate
omega-1 ribonuclease S. japonicum. Wu et al. suggested that S. japonicum eggs produce omega-1 ribonuclease
(Wu et al., 2014). Moreover, gene drives for controlling schistosomiasis can be used. For example, anti-Schistosoma
such as fibrinogen-related proteins, thioster-containing proteins (Maier et al., 2019), or cluster of polymorphic
transmembrane genes (Tennessen et al., 2020) can be inoculated into the ovaries of female O. lorelindoensis to
produce schistosomiasis-resistant snails. As a result, snails will not produce parasites that can infect definite
hosts such as humans and mice. In addition, this technique will not kill intermediate host. To find out the
number of transgenic snails after being released in the field, mathematical ideas can be used. For example, Fn+1
= ([6n – 1] : 1) is useful to find out the number of schistosomiasis-resistant snails and susceptible snails in the
field. With this mathematical idea, I found that F2 progeny had a ratio of five schistosomiasis-resistant snails
to one susceptible snail, for example.
In this study, I report the spread of S. japonicum, species and subspecies of the genus Oncomelania, and
genetic manipulation techniques in the genus Oncomelania for the objectives of this study.
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 25 of 38
2. Materials and methods
I used the nucleotide BLAST and systems of mating approaches in this study. I got a study guide from the
University of California Berkeley (UC Berkeley Library) at https://guides.lib.berkeley.edu/ncbi. I used
nucleotide BLAST for two or more alignments to have such as accession numbers, identity percentage, and
query cover percentage. For query cover, if the first BLAST showed insignificant, I would do BLAST for
somewhat similar sequences (blastn). I do not show E-values and query covers in tables. Data from nucleotide
sequences results were used to draw tree view slanted cladogram of S. japonicum, and the genus Oncomelania
in evolution. In addition, nucleotide BLAST was used to determine the distribution of S. japonicum from China
and Japan to Southeast Asia, and the origin of the genus Oncomelania. The nucleotide BLAST was performed
with the NCBI nucleotide BLAST.
Generation of tree view slanted cladogram was done as follows: First, from the nucleotide NCBI BLAST
results go to Description tab and click the Distance tree of the results links. Second, when the rectangle
cladogram displays: go to the menu Tools > Layout and select Slanted Cladogram.
The CRISPR/Cas9 system and systems of mating were used to find progeny that resistant to S. japonicum.
The CRISPR/Cas9 and anti-Schistosoma was inoculated into female O. lorelindoensis. Systems of mating were
intended to predict total progeny in each generation after transgenic snails are introduced in the field.
2.1. Nucleotide BLAST on S. japonicum
For the nucleotide BLAST approach in S. japonicum, I accessed the reference sequence from GenBank at the
National Center for Biotechnology Information (NCBI). These include the accession numbers:
KU196306.1 (China)
KU196408.1 (West China)
KU196358.1 (Japan)
KU196377.1 (China)
KU196299.1 (China)
KU196362.1 (West China)
KU196398.1 (Taiwan)
KU196379.1 (Philippine)
KU196348.1 (Indonesia)
Except the identity percentages, nucleotide BLAST results were also used to determine the spread of
S. japonicum from China to such as Japan and Indonesia. Tree for relationship in evolution was also shown.
2.2. Nucleotide BLAST on the genus Oncomelania
For the nucleotide BLAST approach in the genus Oncomelania, I accessed the reference sequence from GenBank
at NCBI. These included accession numbers (Attwood et al., 2015; Kameda and Kato, 2011):
AB611791.1 (Japan)
KR002675.1 (West China)
GU367391.1 (China)
KR002674.1 (China)
KR002673.1 (Japan)
DQ112271.1 (Taiwan)
DQ212796.1 (China)
DQ112282.1 (Taiwan)
DQ112287.1 (Philippine)
Except the identity percentage, nucleotide BLAST results were also used to determine the development of the
genus Oncomelania. Tree for relationship of evolutionary was also shown. In this study, I did not find any
information regarding O. lorelindoensis for GenBank accessions.
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 26 of 38
2.3. Creation of transgenic Oncomelania lorelindoensis
S. japonicum can be eliminated by genetic and mathematical approaches. Guadalupe Resistance Complex
(GRC = PTC1) contains resistance to alleles Biomphalaria glabrata. The PTC1 contains a dominant allele, which
confers an 8-fold decrease in infectivity. Both PTC1 and PTC2 suggest a model of interaction via molecular
recognition mediated by TM1 gene polymorphism. The TM1 genes include B and T-Cell receptors, Toll-like
receptors, major histocompatibility complex genes, and similar host defense genes. The TM1 gene often plays
a role in immunological recognition (Maier et al., 2019). Tennessen et al. suggested that polymorphic
transmembrane cluster 2 PTC2 TM1 gene is an obvious candidate to defense against schistosomiasis (Tennessen
et al., 2020). Either PTC1 TM1 or PTC2 TM1 genes can be coupled to a CRISPR-mediated gene drive and spread
through wild-type snails’ population to confer resistance to S. mansoni infection (Maier et al., 2019; and Tennessen
et al., 2020). In this study, I used the clusters of polymorphic transmembrane genes PTC1 TM1 and/or PTC2
TM1 as anti-Schistosomal assuming that these genes exist in the genus Oncomelania.
Elimination of the freshwater snails is not always effective in the long-term. Use of genetic manipulations
would be very helpful for the control of S. japonicum, especially through an intermediate host as the genus
Oncomelania. These techniques can eliminate parasite without killing the snails. However, uses of genetic
manipulations require further investigations before it can work in the field. In this study, I introduced AAV
vector and CRISPR/Cas9 for creation of S. japonicum-resistant transgenic female O. lorelindoensis (Nelwan,
2021a). Although I have not had snails that are resistant to this parasite, even in the laboratory, it is likely to
eliminate this parasite through genetic manipulations. Based on mathematical calculations, the elimination of
schistosomiasis can be done through genetic manipulation in snails. Systems of mating can mathematically
produce schistosomiasis-resistant snails.
The three fundamental requirements in editing with CRISPR/Cas9 include Cas9 endonuclease, single-
guide RNA (sgRNA), and repair template DNA (donor) (Famakinde, 2018). The Cas9 homolog consists of
such as Nme1Cas9 and Nme2Cas9 (Ibraheim et al., 2021). It combs through the genome of the organism and
acts as molecular scissor that cuts a specific DNA sequence at a genomic locus. The sgRNA is designed to
match and target the desired DNA sequence to be deleted. The donor DNA provides a template for genomic
repair of the cleaved locus (Famakinde, 2018). The CRISPR/Cas9 system can tightly hold the anti-schistosomal
donor DNA. Delivery vectors such as AAV and lentivirus can be used as delivery tools in genetic manipulations.
Delivery vectors are packaged into the same virion (Ibraheim et al., 2021).
For a study need, it could be designed a virion package as AAV:Nme2Cas9:sgRNA:PTC1or2TM1. Then,
the package is co-injected into the blastocyst stage embryos of the O. lorelindoensis. Site for the injection of the
entire cassette is in the ovary of the female (Famakinde, 2018; and Nelwan, 2021a) O. lorelindoensis (Nelwan,
2021a). This technique will produce S. japonicum-resistant transgenic snails. If these transgenic snails are
released in the field, snails can produce progeny of schistosomiasis-resistant snails in the next generations
genetically and mathematically.
2.4. Systems of mating for transgenic snails in the field
Systems of mating in the Schistosoma’s intermediate hosts are not the same. The genus Bulinus and the genus
Biomphalaria are hermaphrodites. The genus Bulinus is the intermediate host of S. haematobium. The genus
Biomphalaria is the intermediate host of S. mansoni. As the intermediate host of S. japonicum, the genus Oncomelania
has separate sexes. Therefore, progeny in each generation in the genus Bulinus/Biomphalaria should be not the
same as the genus Oncomelania.
Transgenic snails in the field will follow systems of mating patterns. These systems of mating will include
out-breeding and inbreeding. These are crosses between transgenic S. japonicum-resistant snails and S.
japonicum-susceptibility wild-type snail. These crosses will be in O. lorelindoensis, including crosses between
transgenic, hybrid, and wild type snails. Results will be progeny such as F1, F2, and F3.
3. Results
3.1. Identity percentages of S. japonicum
The nucleotide BLAST results showed that China’s S. japonicum (KU196306.1) shared a similar identity of
99.89% with Indonesia’s S. japonicum (KU196348.1) and 99.87% with the Philippine’s S. jaonicum (KU196379.1).
The sequence of KU196408.1 shared 99.72% identity with KU196348.1 and 99.71% with KU196379.1. The
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 27 of 38
sequence of KU196377.1 shared 99.61% identity with KU196348.1 and 99.60% with KU196379.1. Sequence of
KU196358.1 shared 99.34% identity with KU196348.1 and 99.33% with KU196379.1. Sequence of KU196299.1
shared 99.31% identity with KU196348.1 and 99.30% with KU196348.1 and 99.30% with KU196379.1. Sequence
of KU196362.1 shared 99.29% identity with KU196348.1 and 99.28% with KU196379.1. Sequence of
KU196398.1 shared 98.00% identity with KU196348.1 and 98.01% with KU196379.1 (Table 1 and Figure 1).
Table 1: Percentage identities of Schistosoma japonicum for
China, Japan, Indonesia, Philippine and Taiwan
Indonesia Philippine
Country/region KU196348.1 KU196379.1
KU196306.1 99.89% 99.87%
Mid-Yangtze-THP
KU196408.1 99.72% 99.71%
West
KU196377.1 99.61% 99.60%
Mid- Yangtze-THP
KU196358 99.34% 99.33%
Japan
KU196299.1 99.31% 99.30%
Mid-Yangtze-PLB
KU195362.1 99.29% 99.28%
West
KU196398.1 98.00% 98.01%
Taiwan
Figure 1: BLAST tree view slanted cladogram of Schistosoma japonicum
Note:Indonesia’s Schistosoma japonicum (KU196348.1) and the Philippines’s S. japonicum (KU196379.1) flank China’s S.
japonicum (KU196299.1). The KU196399.1 is close to KU196306.1 that also close to Japan S. japonicum (KU196358.1).
Modified from NCBI BLAST pairwise alignment.
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 28 of 38
The nucleotide BLAST of KU196348.1 (Indonesia) shared 99.87% sequence identity with KU196379.1
(Philippines). All query covers were 100%. These identity percentages suggest that Indonesia’s S. japonicum
derived from China (e.g., 99.89% and 99.72%), Japan (99.34%), or even Taiwan (98%).
3.2. Identity percentages of the genus Oncomelania
Nucleotide BLAST results showed that West China’s O. robertsoni (KR002675.1) shared 87.25% identity with
the Philippines’s O. quadrasi (DQ112287.1), query cover is 93% (not shown in Table 2). Japan’s O. hupensis
Table 2. Percentage identities of genus Oncomelania of China, Japan, Taiwan and the Philippines
Japan
Accession AB611791.1 Country/region
KR002675.1 84 .62 % West China
KR002673.1 82.36% Japan
KR002674.1 82.19% China
DQ112271.1 82.13% Taiwan
DQ112287.1 82.13% Philippine
GU367391.1 81.97% China
DQ212796.1 81.97% China
DQ112282.1 81.97% Taiwan
Japan
Accession KR002673.1 Country/region
DQ112271.1 95.15% Taiwan
GU367391.1 94.81% China
DQ112282.1 94.48% Taiwan
DQ212796.1 94.14% China
KR002674.1 93.97% China
KR002675.1 88 .11 % West China
DQ112287.1 86.26% Philippine
Philippine
Accession DQ112287.1 Country/region
KR002675.1 87 .25 % West China
KR002673.1 86.26% Japan
DQ112271.1 85.58% Taiwan
DQ212796.1 85.11% China
DQ112282.1 84.95% Taiwan
GU367391.1 84.48% China
KR002674.1 84.40% China
AB611791.1 82.13% Japan
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 29 of 38
Table 2 (Cont.)
West China
Accession KR002675.1 Country/region
DQ212796.1 88.80% China
DQ112282.1 88.61% Taiwan
DQ112271.1 88.27% Taiwan
KR002673.1 88.11% Japan
KR002674.1 88.46% China
DQ112287.1 87.25% Philippine
China
Accession GU367391.1 Country/region
KR002674.1 98.66% China
DQ212796.1 97.96% China
DQ112282.1 97.65% Taiwan
DQ112271.1 97.49% Taiwan
KR002673.1 94.81% Japan
KR002675.1 88 .29 % West China
DQ112287.1 84.48% Philippine
Note:PLB: Poyang Lake Basin, China; THP: Mid-Yangtze to Lower Yangtze Taihu, Plain, China.
nosophora (KR002673.1) shared 86.26% identity with DQ112287.1. Taiwan’s O. hupensis chiui (DQ112271.1)
shared 85.58% identity with DQ112287.1. Taiwan’s O. hupensis formosana (DQ112282.1) shared 84.95% identity
with DQ112287.1. China’s O. hupensis hupensis (KR002674.1) shared 84.40% identity with DQ112287.1 (Table 2).
Finally, DQ112287.1 shared 82.13% identity with Japan’s O. minima (AB611791.1) (Table 2 and Figure 2).
Figure 2: BLAST tree view slanted cladogram of the genus Oncomelania
Not e: Southeast Asia’s Oncomelania was close to Oncomelania robertsoni. Modified from NCBI BLAST pairwise alignment.
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 30 of 38
Table 3: The genus Oncomelania
Species O. hupensis O. robersoni O. minima
O. h. hupensis 98.66% 88.46% 82.19%
O.h. tangi 97.96% 88.80% 81.97%
O. h. formosana 97.65% 88.61% 81.97%
O. h. chiui 97.49% 88.27% 82.13%
O. h. nosophora 94.81% 88.11% 82.36%
O. quadrasi 84.48% 87.25% 82.13%
I did not find O. lorelindoensis in GenBank for accession. As a result, the nucleotide sequence could not
be done.
The nucleotide sequence results showed that O. hupensis developed O. hupensis hupensis, O. hupensis tangi,
O. hupensis formosana, O. hupensis nosophora, and O. hupensis chiui. In addition, sequence results showed that O.
quadrasi was closer to O. robertsoni than to O. hupensis (Table 3).
3.3. Genetic biocontrol of Schistotomiasis japonica
The creation of transgenic female O. lorelindoensis resulted in transgenic female O. lorelindoensis (Figure 3).
Then, transgenic female O. lorelindoensis snails were released into the field containing schistosomiasis-
Figure 3: Transgenic snails
Not e: Creation transgenic snails via clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system in
a snail vector schistosome. DSB, double-strand break; HDR, homology-directed repair; PCR, polymerase chain
reaction; RE, restriction enzyme (Modified from Famakinde, 2018). (Oncomelania snail image taken from Gordon
et al., 2019).
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 31 of 38
susceptible O. lorelindoensis snails. Crossing results between transgenic snails and schistosomiasis-susceptible
wild-type snails resulted in as described in Figure 4. For example, transgenic snails crossed with shistosomiasis-
Figure 4: Transgenic snails in the field
Not e: Transgenic snails for field control of schistosomiasis japonica transmission. Transgenic snails released in the field
resulted in a ratio of two schistosomiasis-resistant snails to one susceptible snail in F1, a ratio of five schistosomiasis-
resistant snails to one susceptible snail in F2, and a ratio of 35 resistant-resistant snails to one susceptible snail in F3.
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 32 of 38
Figure 4 (Cont.)
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 33 of 38
Figure 4 (Cont.)
F3 progeny: thirty-five resistant and one susceptible.
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 34 of 38
susceptible snails resulted in a ratio of two schistosomiasis-resistant snails to one susceptible snail in
F1 progeny and total was F1 = 31 = 3 snails. In F2, it resulted in a ratio of five schistosomiasis-resistant snails to
one susceptible snail (Fn + 1 = ([6n – 1] : 1)) and total was F2 = 61 = 6 snails. These results resulted in mathematical
ideas for F1 is ([3n – 1] : 1) in which n is 1 and the mathematical idea for Fn+1 2 is ([6n – 1] : 1) in which n is
natural number. Total F1 = 3n and total Fn+1 = 6n.
4. Discussion
For S. japonicum, lineages from Southeast Asia were in Haplogroup A. S. japonicum from the lake regions of
China in Haplogroup A1a migrated to Southeast Asia at ~3,000-4,000 years ago. It migrated to mountainous
regions at ~ 5,000 years ago. The data suggest that S. japonicum originated from the lake area of China, with the
parasite spreading to Japan around 7,000 years ago. It radiated into the mountains of China about 5,000 years
ago, and to the Philippines and Indonesia about 4,000 years ago (Yin et al., 2015).
According to sequence results, I found that Sulawesi’s S. japonicum and the Philippines’s S. japonicum
shared identity percentages almost the same as China’s S. japonicum (99.89% and.99.87%), West China (99.72%
and 99.71%), Japan (99.34% and 99.33%), and West China (99.29% and 99.28%) (Table 1). In addition, Sulawesi’s
S. japonicum was close to the Philippine’s S. japonicum (KU196379.1), China (KU196306.1), Japan (KU196358.10,
China (KU19299.1), and West China (KU196408.1) (Figure 1). This sequence suggests that Southeast Asia’s S.
japonicum originated from Japan and China. Interestingly, Sulawesi’s S. japonicum had a higher identity
percentage than with the Philippines’s S. japonicum (Table 1). This suggests that the spread of S. japonicum
from either Japan or China to Southeast Asia first reached Sulawesi and then to the Philippines. However, if
Sulawesi’s S. japonicum derived from Taiwan, it first reached the Philippines and then Sulawesi, Indonesia.
These results show that Sulawesi’s S. japonicum and the Philippines’s S. japonicum could originate from
China’s ML of the Yangtze River, West China, Japan, or even Taiwan. It did not originate from the ML of the
Yangtze River only, indicating that my finding is novel.
Using a molecular clock, the introduction of O. hupensis across mainland China has been dated to the early
Miocene (ca 22 million years ago), with a high rate of cladogenesis 8-2 million years ago. It was due to the
unusually warm and humid climate of the region at that time and the tectonic turmoil in Japan. The divergence
of the S. japonicum clade has been dated at 4.6 million years ago. This implies that the radiation of O. hupensis
occurred before that of S. japonicum. If the radiation of the snails and worms is heterochronous, there is no
opportunity for coevolution. The implication is also that the ancestral intermediate host differed from those of
the present, which again makes coevolution unlikely. For the genus Oncomelania dispersal, the ML reaches of
the Yangtze River clade brought out the Fujian coastal plain (China), Japan, Taiwan, and the Philippines
populations of O. hupensis hupensis. Such a re-colonization of Japan by mainland Chinese O. hupensis is
consistent with the “East to West” hypothesis. This hypothesis proposes that Oncomelania was from Australia
and via the Philippines, according to this hypothesis, after reaching Japan; Proto-Oncomelania gives rise to O.
hupensis in mainland China. Oncomelania hupensis (KR002674.1) re-colonized Japan (KR002673.1), and spread
to West China (KR002675.1). Oncomelania hupensis then radiated to Taiwan, the Philippines and Sulawesi
(Attwood et al., 2015).
According to the sequence results using the NCBI BLAST tool, I found that the Philippines’s O. quadrasi
(Garcia et al., 1980) (DQ112287.1) shared an identity of 87.25% with West China’s O. robertsoni (KR002675.1).
It is the highest percentage. The second rank was Japan’s O. hupensis nosophora (KR002673.1) with an identity
of 86.26%. The third rank was Taiwan’s O. hupensis chiui (DQ112271.1) with an identity of 85.58%. China’s
Oncomelania hupensis (GU367391.1) shared an identity of 84.48% with O. quadrasi (Table 2). These results
suggest that O. quadrasi is close to O. robertsoni, O. hupensis nosophora, and O. hupensis chiui, especially O.
robertsoni. Oncomelania hupensis nosophora did not originate from O. minima. It is closer to O. hupensis with an
identity of 94.81% compared to O. minima with an identity of 82.36 % (Table 2). Thus, O. hupensis nosophora
originated from O. hupensis, not from O. minima. Sequences results confirmed that O. hupensis gave rise to O.
hupensis tangi, O. hupensis chiui, O. hupensis formosana, and O. hupensis nosophora. Oncomelania hupensis differs
from O. quadrasi (Table 3), suggesting O. quadrasi did not originate from O. hupensis as it is too distance.
Oncomelania quadrasi is closer to O. robertsoni than to O. hupensis (Table 2 and Table 3). Therefore, O. robertsoni
must be a complete species, along with O. lorelindoensis and O. quadrasi. These three species are beyond O.
hupensis group and O. minima (Figure 2). Oncomelania hupensis lindoensis should be O. lorelindoensis as it drives
from the Lindu Valley and the Lore sub districts (the Bada Valley and the Napu Valley). The Lindu Valley is
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 35 of 38
within the Lore Lindu National Park. Oncomelania hupensis quadrasi must be O. quadrasi as suggested by
Woodruff et al. (Garcia et al., 1980) and beyond the O. hupensis group. Finally, O. hupensis robersoni must be O.
robertsoni (Figures 2 and 5).
In Central Sulawesi, the endemic areas of S. japonicum include the Bada Valley, the Lindu Valley, and the
Napu Valley (Budiono et al., 2019; and Samarang et al., 2018). Carney et al. found the intermediate host of
Sulawesi’s S. japonicum in 1971 in the Lindu Valley (Nelwan, 2021c). Based on sequence results and added
with O. lorelindoensis, I found that the genus Oncomelania consist of five species and five subspecies (Table 4 and
Figure 5). It is new findings in the genus Oncomelania, especially O. lorelinduensis in Central Sulawesi.
Table 4: The genus Oncomelania
Name Country/region
O. hupensis China
O. h. chiui Taiwan
O.h. formosana Taiwan
O. h. hupensis China
O. h. nosophora Japan
O. h. tangi China
O. lorelindoensis Indonesia
O. minima Japan
O. robertsoni China
O. quadrasi Philippine
Note: O: Oncomelania and O.h: Oncomelania, Hupensis
Figure 5: The genus Oncomelania
Not e: Five species of the genus Oncomelania: O. minima (1), O. robertsoni (2), O. hupensis (3), O. quadrasi (4), and O.
lorelindoensis (5).
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 36 of 38
It could be likely that Sulawesi’s S. japonicum and O. lorelindoensis has spread to other areas of the region.
Control of this parasite in Sulawesi can help prevent the spread of the parasite in the region. It can be through
genetic manipulation in the intermediate host (Nelwan, 2021b).
Genetic manipulation techniques can help control schistosomiasis, especially schistosomiasis japonica in
Central Sulawesi, Indonesia (Nelwan, 2021a). Indonesia has made great efforts to eliminate this parasite
(Gordon et al., 2019). However, schistosomiasis still exists and has even expanded to new areas around
endemic areas of the parasite (Nurwidayati et al., 2018; and Samarang et al., 2018). To support the elimination
of this parasite, genetic manipulations in intermediate host O. lorelindoensis can be done. Concepts of creation
for transgenic female snails and systems of mating have been available. Based on those concepts, I found that
resistant snails has a ratio of 35 schistosomiasis-resistant snails to one susceptible snail in F3 progeny (F2+1 =
([62 – 1] : 1) (Figure 4). Unfortunately, genetic manipulation in snails has ethical challenges, and these must be
discussed among such scientists, politicians, and relevant communities (Nelwan, 2021a; and Maier et al.,
2019).
The National Academies of Science, Engineering, and Medicine (NASEM) have emphasized the importance
of an interdisciplinary perspective on gene drive research. It explicitly attends to complex human values and
the necessity of the community, stakeholders, and public engagement to accompany technical research and
development. Although decision-making involves risk assessment, the prevailing uncertainties of genome
engineering technology in snails, other organisms, and its behavior in the wild prevent appropriate risk/
benefit analysis. Therefore, some have emphasized the need to allow sufficient time to develop amendments to
current regulatory framework (Maier et al., 2019).
There are two limitations of the study. First, critic says that it is likely that sequences with BLASTN
techniques have a lower level confidence than with phylogenetic tree analysis techniques. However, it should
be known that both BLASTN and phylogenetic tree analysis use nucleotide of DNA/RNA to have tree views
in evolutionary. Thus, it should be no different between BLASTN and phylogenetic techniques I think. Second,
there is no information in details about CRISPR/Cas9 editing. For example, total snails that would be used in
the study were not mentioned.
Detection of schistosomiasis plays an important role in elimination efforts of schistosomiasis. Several
detection tools are available: microscopic, serological, molecular, and imagine techniques. For example,
ultrasonography technique can detect S. japonicum worms in mice (Maezawa et al., 2018). Therefore, this tool
can help detect a status of schistosomiasis in an endemic area before or after treatment with genetic biocontrol
of schistosomiasis. Ultasonography can also detect this disease in humans (Figure 6).
5. Conclusion
Sulawesi’s S. japonicum originated from Japan and China. Its intermediate host is O. lorelindoensis. Oncomelania
lorelindoensis is close to O. quadrasi. The genus Oncomelania consists of five species and five subspecies. Genetic
manipulation techniques can help for eliminating schistosomiasis, especially schistosomiasis japonica. These
techniques can include AAV vectors and CRISPR/Cas9 system. Based on systems of mating patterns, the
number of progeny in each generation can be counted by mathematical ideas. Genetic manipulation technique
does not kill snails. However, before use of this technique, ethical issues must carefully be considered.
Figure 6: Hepatosplenic schistosomiasis in human
Not e: A and B images of liver showing hepatic schistosomiasis. C shows enlarge spleen with dilated spenic vein (Taken
from Sah et al., 2015).
Martin L. Nelwan / Afr.J.Bio.Sc. 4(4) (2022) 23-38 Page 37 of 38
6. Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-
profit factors.
Acknowledgment
Exclusively, M. Nelwan performed research and manuscript development.
Conflicts of interest
I declare that no conflicts of interest exist.
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Cite this article as: Martin L. Nelwan (2022). Indonesia Schistosoma japonicum: Origin, genus oncomelania,
and elimination of the parasite with cluster genes inoculated into female Oncomelania lorelindoensis via
CRISPR/Cas9 system. African Journal of Biological Sciences. 4(4), 23-38. doi: 10.33472/AFJBS.4.4.2022.23-38.
