Abstract
Background
Ascaris lumbricoides causes human ascariasis, the most prevalent
helminth disease, infecting approximately 1 billion individuals
globally. In 2019 the global disease burden was estimated to be 754,000
DALYs and resulted in 2090 deaths. In the absence of a vaccination
strategy, treatment of ascariasis has relied on anthelminthic
chemotherapy, but drug resistance is a concern. The propensity for
reinfection is also a major challenge to disease control; female worms
lay up to 200,000 eggs daily, which contaminate surrounding
environments and remain viable for years, resulting in high
transmission rates. Understanding the molecular mechanisms of
reproductive processes, including control of egg production,
spermatogenesis, oogenesis and embryogenesis, will drive the
development of new drugs and/or vaccine targets for future ascariasis
control.
Methods
Transcriptome profiles of discrete reproductive and somatic tissue
samples were generated from adult male and female worms using Illumina
HiSeq with 2 × 150 bp paired-end sequencing. Male tissues included:
testis germinal zone, testis part of vas deferens, seminal vesicle and
somatic tissue. Female tissues included: ovary germinal zone, ovary
part of the oviduct, uterus and somatic tissue. Differentially
expressed genes (DEGs) were identified from the fragments per kilobases
per million reads (FPKM) profiles. Hierarchical analysis was performed
to identify tissue-specific genes. Furthermore, Gene Ontology (GO) and
Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed
to identify significant terms and pathways for the DEGs.
Results
DEGs involved in protein phosphorylation and adhesion molecules were
indicated to play a crucial role in spermatogenesis and fertilization,
respectively. Those genes associated with the G-protein-coupled
receptor (GPCR) signaling pathway and small GTPase-mediated signal
transduction pathway play an essential role in cytoskeleton
organization during oogenesis. Additionally, DEGs associated with the
SMA genes and TGF-β signaling pathway are crucial in adult female
embryogenesis. Some genes associated with particular biological
processes and pathways that were identified in this study have been
linked to defects in germline development, embryogenesis and
reproductive behavior. In the enriched KEGG pathway analysis, Hippo
signaling, oxytocin signaling and tight junction pathways were
identified to play a role in Ascaris male and female reproductive
systems.
Conclusions
This study has provided comprehensive transcriptome profiles of
discrete A. lumbricoides reproductive tissue samples, revealing the
molecular basis of these functionally important tissues. The data
generated from this study will provide fundamental knowledge on the
reproductive biology of Ascaris and will inform future target
identification for anti-ascariasis drugs and/or vaccines.
Graphical Abstract
[41]graphic file with name 13071_2022_5602_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s13071-022-05602-2.
Keyword: Ascaris lumbricoides, Gene expression, Reproductive tissue,
RNA-Seq, Transcriptome, Transcriptomics
Background
Ascaris lumbricoides is a soil-transmitted helminth (STH) that cause
human ascariasis, especially in tropical and subtropical regions of the
world. Ascariasis has the highest prevalence of all helminthiases,
infecting approximately 0.8–1.2 billion individuals worldwide,
especially preschool and school-aged children [[42]1]. The reproductive
capacity of Ascaris drives the life cycle and exacerbates disease
transmission where each individual female can produce > 200,000
fertilized eggs daily [[43]2]. When the eggs are deposited and
incubated in the soil, they develop into an infective stage
(embryonated egg), which can remain viable for several years despite
extreme environmental conditions that may include periods of extreme
temperature fluctuations and drought; Ascaris eggs are also resistant
to chemical treatment [[44]3]. Control is primarily based on delivery
of anthelminthic therapy to symptomatic patients or periodic mass drug
administration in endemic areas. There is currently no vaccine for
human ascariasis. Although benzimidazoles are effective in treating
ascariasis, the development of drug resistance is a concern
[[45]4–[46]6]. As a result, sustainable future control of ascariasis
will rely on the identification of new molecular targets. Understanding
the molecular mechanisms of egg production and spermatogenesis in
female and male A. lumbricoides will contribute to the discovery of
novel drug and vaccine candidates. Targeting these tissues will limit
the large number of eggs produced.
Tissue-specific transcriptomics offers an opportunity to identify gene
targets that underlie key functions at the tissue and cellular level.
Transcriptome profiles of several helminths, generated using microarray
or RNA sequencing (RNA-Seq), have targeted specific tissues
[[47]7–[48]10], stages [[49]11–[50]13] and sexes [[51]14, [52]15].
Indeed, transcriptome profiles of both germline and somatic tissues of
the pig roundworm Ascaris suum, a closely related species to A.
lumbricoides, have been generated [[53]16]. In addition to
transcriptomics, the microRNA profiles of male and female A. suum
reproductive tissue samples have also been analyzed, focusing on the
regulation of gene expression [[54]17–[55]19]. Despite this, there are
no equivalent datasets for A. lumbricoides, which would enable
comparative analyses of the molecular mechanisms and gene expression
profiles between the two Ascaris species. As such, transcriptome
analysis of A. lumbricoides reproductive tissue samples will complete
the molecular basis of the genus Ascaris, which may aid in the
establishment of effective control of both related species that pose
zoonotic and anthroponotic threats [[56]20].
The large worm size and the linear structure of the reproductive tissue
in A. lumbricoides allows for the dissection and isolation of the
discrete stages of gametogenesis in both male and female worms. This
study employs RNA-Seq to generate transcriptome profiles of discrete
reproductive and somatic tissue samples in adult male and female A.
lumbricoides to identify significant differentially expressed genes
(DEGs) that can be attributed to roles in egg production and
spermatogenesis. Data generated in this study will contribute to better
understanding of the molecular mechanisms involved in A. lumbricoides
egg development and spermatogenesis. These insights will inform future
development of target-specific ascariasis control strategies in humans,
and potentially in pigs.
Methods
Study area
The study site was located in the Ban Mae Salid Luang village, Mae Song
Sub-District, Thasongyang District and Tak Province close to the
Thai-Myanmar border. This study was approved by the Human Research
Ethics Committee of the Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand (MUTM 2021–020-01 and MUTM 2021–020-02).
Stool examination
With their assent and consent, a total of 186 participants with ages
ranging from 7 to 17 years were recruited to provide stool samples for
the investigation of A. lumbricoides infection. Participants received
labeled sample containers to collect their stools and send them to the
study site for stool examination. Along with collecting stool samples,
data such as age, gender and address were also collected.
All human stool samples were subjected to the Kato-Katz method as
previously reported [[57]21–[58]23]. In brief, a stainless-steel sieve
(size 40 mesh: 420 µm sieve opening) was used to press individual stool
samples, and the non-retained material was used to fill a kit template
with 39.2 mg material. The sample was covered with glycerin-malachite
green-soaked cellophane and firmly pressed to disseminate the stool
across the surface. After 30 min, the slide was examined under a light
microscope. Ascaris lumbricoides eggs were recorded. All samples were
anonymized.
When utilizing the Kato-Katz method, 32 of the 186 participants had A.
lumbricoides-positive stools. Ascaris lumbricoides-infected
participants were treated orally with a single dose of albendazole
(400 mg) according to WHO guidelines [[59]24]. The medical treatment
was delivered under the physical examination and supervision of a
medical doctor individually. Patients were monitored for 1 h at the
study site for the appearance of any side effects from the medication,
such as nausea, vomiting, headaches and dizziness. After the
participant returned home, they continued to monitor any side effects
on their own, and if any were found, they promptly received medical
attention. No patients presented any side effects at any stage. The
results of the stool examination were reported to the local
health-promoting hospitals for further investigation and action on the
prevention and control strategy.
Collection of adult A. lumbricoides for RNA-Seq
A total of 32 individuals with A. lumbricoides-positive stool eggs were
approached for treatment, and whole stools were collected daily for
4 days with their assent and consent. Adult A. lumbricoides were
obtained from participants after albendazole treatment; infected
individuals collected whole stool in plastic garbage bags daily for
4 days post-albendazole administration. Since worms could be expelled
at different times from each participant, the stool was collected
immediately after defecation and transferred to the field laboratory
station within 3 h. At the field laboratory station, adult worms were
recovered from the stool sample and rinsed several times with sterile
0.85% normal saline solution (NSS) to remove fecal contamination.
According to the worm expulsion, each adult worm collected was examined
to ensure its vitality; 15 of the 39 individual worms that were
retrieved were still viable. To obtain the best quality adult worm for
RNA isolation, only worms that were viable and in good physical
condition were collected. Subsequently, male and female worms were
distinguished morphologically under a stereomicroscope. Three adult
male and three adult female worms of good quality, which were retrieved
from different participants, were employed in the experiment. Male and
female worms were then dissected under a stereomicroscope to retrieve
their somatic and reproductive tissues. Body wall muscles at the
anterior region were used as somatic tissue samples. The reproductive
tissue samples were completely excised and placed in a Petri dish
containing NSS. Four distinct regions with the male reproductive
tissues were dissected: (i) the germinal zone of the testis (Tg),
approximately 5 cm in length from the tip of the anterior testis; (ii)
testis, part of vas deferens (Tv); (iii) seminal vesicle (Sv); (iv)
male somatic tissue (Stm). Four distinction regions with the female
reproductive tissue were dissected: (i) the germinal zone of the ovary
(Og); (ii) ovary, part of the oviduct (Ov); (iii) uterus (Ut); (iv)
female somatic tissue (Stf). The dissection of somatic and reproductive
tissues is shown in Additional file [60]1: Figure S1. Note that the
distinct regions within the male and female reproductive tracts, as
outlined above, were identified based on a previous publication
[[61]18]. Dissected tissue samples were immediately frozen on dry ice
and transported back to the main laboratory in Bangkok, Thailand, where
they were stored at − 80 °C prior to RNA extraction. Six worms in
total, three distinct male worms and three distinct female worms, were
used in this study. Each worm was dissected for four different tissue
types for examination. Therefore, RNA sequencing was performed on a
total of 24 tissue samples. Each worm was taken from a different
participant.
Total RNA extraction, library construction and sequencing
Total RNA was isolated from each sample using TRIzol Reagent according
to the manufacturer's instructions (Thermo Fisher Scientific, Waltham,
MA, USA). The quantity and quality of total RNA was determined using an
Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA),
NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel
electrophoresis. One microgram of total RNA with RIN value > 6.5, which
met the quality requirements of the manufacturer, was processed for
library preparation and RNA sequencing by Vishuo Biomedical (Thailand)
Ltd. Briefly, next-generation sequencing libraries were prepared
according to the manufacturer’s protocol [NEBNext^® Ultra™ RNA Library
Prep Kit for Illumina^®, New England Biolabs (NEB), Ipswich, MA, USA].
Poly(A) mRNA isolation was performed using the Poly(A) mRNA Magnetic
Isolation Module (NEB). mRNA fragmentation and priming were performed
using First-Strand Synthesis Reaction Buffer and random primers (NEB).
First-strand cDNA was synthesized using ProtoScript II Reverse
Transcriptase, and second-strand cDNA was synthesized using
Second-Strand Synthesis Enzyme Mix. Following this, the purified
double-stranded cDNA was treated with End Prep Enzyme Mix to repair
both ends and add a dA-tailing in one reaction, followed by a T-A
ligation to add adaptors to both ends. Next, size selection of
adaptor-ligated DNA was performed using VAHTS DNA clean beads (Vazyme
Biotech Co., Ltd, Nanjing, China), and fragments of approximately
420 bp (with an approximate insert size of 300 bp) were recovered. Each
sample was then amplified by PCR for 13 cycles using P5 and P7 primers
(see Additional file [62]2: Table S1), with both primers carrying
sequences that can anneal with flow cells to perform bridge PCR and the
P7 primer carrying a six-base index that allows for multiplexing. The
PCR products were cleaned up using VAHTS DNA clean beads, validated
using Qsep 100 (Bioptic, Taiwan, China) and quantified using a Qubit
3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Subsequently,
libraries with different indices were multiplexed and loaded on an
Illumina HiSeq instrument according to the manufacturer’s instructions
(Illumina, San Diego, CA, USA). Sequencing was performed using a
2 × 150-bp paired-end configuration, with 6.0-Gb raw data per sample.
Image analysis and base calling were conducted using the HiSeq Control
Software (HCS) + OLB + GAPipeline-1.6 (Illumina).
Bioinformatics analysis
Bcl2fastq (v.2.17.1.14) was used to process the original image data for
base calling and preliminary quality analysis. The quality assessment
of the sequencing data was performed using FastQC (v.0.10.1) [[63]25].
The base quality scores, expressed in Q Phred. Cutadapt (v.1.9.1), were
used for data filtering to remove the adapter sequences, 5′ or 3′ end
bases containing N’s or quality values < 20, and reads that were <
75 bp long after trimming [[64]26]. Subsequently, filtered data were
aligned to the reference A. lumbricoides genome (WormBase ParaSite,
BioProject PRJEB4950, Taxonomy ID 6252). Short-read alignment was
performed using Hisat2 (v.2.0.1) with default parameters [[65]27].
Gene differential expression analysis
Read density was used to calculate the level of gene expression of all
genes. Based on the read counts from HT-seq (v.0.6.1), fragments per
kilobases per million reads (FPKM) were used to calculate gene
expression using the formula outlined below [[66]28]. Since three
biological tissue replicates were performed, Pearson’s correlation was
used to calculate the correlation of gene expression between samples to
assess RNA-Seq quality. Principal component analysis (PCA) was also
used to assess the correlation of gene expression between samples to
reduce data complexity.
[MATH: FPKM=TotalExonfragments(MappedreadsMillions×Exonlengthkb) :MATH]
To identify the DEGs of the discrete reproductive tissue samples, the
gene expression level of all genes (FPKM profiles) of each discrete
reproductive tissue was compared across the tissue samples using the
Bioconductor package DESeq2 (v.1.6.3) [[67]29]. Subsequently,
significant DEGs were identified based on fold change > 2 and Q-value
(FDR, P-adj) < 0.05. Normalized FPKM were hierarchically clustered to
classify DEGs with similar expression patterns using Pearson’s
correlation. Clustered data were graphically depicted (heatmap) using
gplots in the R package.
Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
enrichment analyses
The annotation was carried out using the gene classification system,
Gene Ontology (GO) database. GO annotation provides a set of
dynamically updated standard vocabulary to describe the properties of
genes and gene products in the organism. GO contains three ontologies
that describe the molecular function, cellular component and biological
process of the gene. To identify the GO terms that were enriched among
DEGs against the transcriptomic background, GO enrichment analysis was
performed using GOSeq (v.1.34.1) [[68]30], based on an extension of the
hypergeometric distribution with a threshold for filtering
overrepresented P-value
[MATH: ≤ :MATH]
0.05.
Kyoto Encyclopedia of Genes and Genomes (KEGG) is the primary public
pathway database used in this analysis [[69]31]. Pathway enrichment
analysis performed in this section is based on KEGG pathway units and
used a hypergeometric test, with a threshold Q-value of
[MATH: ≤ :MATH]
0.05, to identify the pathways of the DEGs that are significantly
enriched against the transcriptome background [[70]32]. The ratio of
the number of genes differentially expressed in the pathway to the
total number of genes in the pathway (rich factor), Q-value and the
number of genes enriched in the pathway were used to assess the degree
of KEGG enrichment.
Validation of gene expression by RT-qPCR
The top DEGs specific for each cluster were selected based on top three
normalized expression levels within each cluster to validate the
RNA-Seq results. RT-qPCR was used as follows. A DEG was selected from
Tg, Sv and Ut tissue samples, which are exclusively indicated in
Clusters C, D and E, respectively. If genes were expressed in groups of
tissues, a DEG was selected primarily from the dominantly expressed
tissue as follows: a DEG was selected primarily from Stf, Tv, Og and Ov
tissue samples as predominantly expressed in the Stf/Stm
tissue-specific (Cluster A), Tv/Tg—tissue-specific (Cluster B),
Og/Ov—tissue-specific (Cluster F) and Ov/Og—tissue-specific (Cluster
G), respectively.
All tissue samples with three biological replicates from the same
samples utilized in RNA-Seq experiments were used as templates to
perform RT-qPCR validation assays for each representative
differentially expressed tissue-specific gene. In summary for RT-qPCR,
initially, first-strand cDNA was synthesized as follows; total RNA
(1 μg) from each tissue sample was treated with 1 U of DNase I (Thermo
Fisher Scientific) before synthesizing first-strand cDNA using a
RevertAid First-Strand cDNA Synthesis kit (Thermo Fisher Scientific)
according to the manufacturer’s instructions. Each 20 μL reaction
mixture contained 1 µg of total RNA, 1 mM of each dNTP and 10 μM Oligo
(dT) primer (Sigma-Aldrich, Inc., Saint Louis, MO). The reaction
mixture was chilled on ice for 1 min after being incubated at 65 °C for
5 min. The reaction mixture was mixed with 4 μl of 5 × RT buffer and 1
μl of Revert Aid RT, and it was then incubated at 42 °C for 1 h.
Afterwards, the reaction mixture was incubated for 5 min at 70 °C.
Second, first-strand cDNA was utilized as the template for the SYBR
Green RT-qPCR. Each 20 μl reaction mixture contained 2 μl of
first-strand cDNA, 1 × iTaq Universal SYBR Green Supermix (Bio-Rad
Laboratories, Philadelphia, PA, USA) and 300 nM of each forward and
reverse primer. Lastly, amplification was carried out using a CFX96
Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA)
according to the following protocol: pre-incubation at 95 °C for 5 min,
followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min. Melting
curve analysis was performed from 65 °C to 95 °C.
Eukaryotic translation initiation factor 6 (eIF6) and NADH cytochrome
b5 reductase were used as internal controls to normalize gene
expression levels [[71]2, [72]7]. The relative gene expression levels
were calculated using the 2^−∆∆Ct method [[73]33, [74]34]. RT-qPCR
experiments were performed in triplicate. Primer sequences for each
target gene were designed using Primer3Plus
([75]http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi)
with default parameters (see Additional file [76]2: Table S1). Prior to
further investigation, each primer pair was verified for specificity by
blasting it against the A. lumbricodes transcriptome database to
identify any non-target transcripts
([77]https://parasite.wormbase.org/Ascaris_lumbricoides_prjeb4950/Info/
Index/).
Results and discussion
RNA sequencing data and quality assessment of discrete A. lumbricoides
reproductive tissues
After quality assessment and trimming, sequences ranging from 40 to 50
million pair-end 150-bp reads were generated from biological replicates
of each tissue sample. As a result, pooled technical replicates of each
tissue sample yielded over 120 million reads, of which more than 98.0%
of total raw reads passed the quality filter. Furthermore,
approximately 97% and 92% of total nucleotide bases had Phred quality
scores > 20 and 30, respectively, indicating high read quality; GC
content in the clean reads ranged from 46 to 49% (Additional file
[78]3: Table S2).
More than 75% of all clean reads from almost all tissue samples were
successfully mapped to the A. lumbricoides reference genome, indicating
that no contamination had occurred and the appropriate reference genome
was selected. Note that one replicate of A. lumbricoides Ut tissue
sample generated a clean read that mapped to the reference genome at a
lower rate than the other tissues and/or replicates, at approximately
65%, which could be explained by variation in tissue samples.
Consistent variation in gene expression profiles among tissue samples
would require further investigation, such as analyzing more samples
from the same tissues/regions within a tissue and/or generating
additional reference genomes. The mappable reads, which ranged from 58
to 81% of the total clean reads, were uniquely mapped to one location
within the A. lumbricoides reference genome, whereas the remaining
reads were mapped to multiple locations within the genome (see
Additional file [79]4: Table S3 for raw, filtered and alignment data
statistics). Further analysis of the distribution of the mappable reads
within the reference genome revealed that > 80% of reads were mapped to
the exon region in all tissue samples whereas the remaining reads were
distributed in the intergenic and intron regions (Additional file
[80]5: Figure S2).
Pearson’s correlation analysis was used to assess RNA-Seq quality
(Additional file [81]6: Figure S3). The results indicate that most
tissue samples displayed reproducibility and consistent quality across
biological replicates, but Ut tissue samples had the lowest congruence
within replicates, which, as highlighted above, could be due to the
tissue-specific variation within samples. On the basis of the PCA, the
sample was divided into six groups, including (i) Tg tissue, (ii) Tv
tissue, (iii) Sv tissue, (iv) two replicates of Ut tissue, (v) Og, Ov
and one replicate of Ut tissue and (vi) Stf and Stm tissue samples
(Fig. [82]1). There was clearly a distinct grouping for male
reproductive tissue samples. However, Og and Ov appeared to be grouped
together for female reproductive tissue samples, with one replicate of
Ut tissue merging in this group, suggesting that they were closely
related to the Og and Ov tissue samples. This might be due to an
immature female adult used in this study, in which the reproductive
organs, especially the Ut tissue, had not fully developed. In addition,
the somatic tissue samples of the male and female worms were grouped
together, demonstrating a high level of correlation between the
transcriptome of the samples (see Additional file [83]6: Figure S3 for
data on correlation between samples).
Fig. 1.
[84]Fig. 1
[85]Open in a new tab
Principal component analysis (PCA) showed congruence within the
replicates of the different samples (replicates for each sample are
indicated by the same color), except one sample of Ut tissue. Male
tissue samples include Tg: the germinal zone of the testis; Tv: testis,
part of vas deferens; Sv: seminal vesicle; Stm: male somatic tissues.
Female tissue samples include Og: the germinal zone of the ovary; Ov:
ovary, part of oviduct; Ut: uterus; Stf: female somatic tissues. Three
biological replicates of each male and female tissue type were analyzed
in this study, with each replicate from a worm derived from a different
participant
However, the limitations based on the acquisition of clinical material
do not make this surprising. Particularly the application of
anthelmintic treatment of individuals and the clearance of adult worms
limited the quality of isolated RNA. We highlight that the value of our
unique clinical data and samples needs to be considered in the context
of our human ethical requirements.
Determination of DEGs across the discrete tissue samples
According to a Venn diagram, analysis of expression indicated overlap
in expression (3147 DEGs) among three male reproductive tissues
(Fig. [86]2). Genes associated with protein phosphorylation, such as
protein kinase domain-containing proteins and serine/threonine-protein
phosphatases, had overlapped expression in male reproductive tissues.
In two tissue comparisons, 3963 (Tg and Tv), 926 (Tg and Sv) and 426
DEGs (Tv and Sv) had expression overlap, while only Tg, Tv and Sv
tissues were discovered to particularly express 1570, 2003 and 1062
DEGs, respectively. All female reproductive tissues showed overlapping
expression of 3534 DEGs. There were 3661 (Og and Ov), 247 (Og and Ut)
and 328 DEGs (Ov and Ut) that had expression overlap in the two tissue
comparisons. It was discovered that only Og, Ov and Ut tissues
specifically expressed 1201, 1127 and 556 DEGs, respectively. The
complete list of DEGs is shown in Additional file [87]7: Table S4. It
is interesting to note that DEGs associated with vesicle-mediated
transport, such as the protein transport protein SEC23, were discovered
to be particularly expressed in the Tv tissue. This protein is the core
component of the coat protein complex II (COPII) and functions to
transport newly synthesized proteins and lipids from the endoplasmic
reticulum (ER) to the Golgi apparatus in cells for secretion
(anterograde transport) [[88]35]. Moreover, DEGs (ALUE 0000599901)
associated with COPI (retrograde transport) were discovered to be
expressed in the Tv tissue, indicating that this tissue participates in
both anterograde and retrograde transport. However, Sv tissue only
displayed retrograde transport activity since only the expressed DEGs
(ALUE 0000977901 and ALUE 0001963101) were linked to COPI. According to
a report, COPI and COPII are both involved in transport of vesicles
during acrosome development [[89]36], suggesting they may be important
for Ascaris spermatogenesis.
Fig. 2.
[90]Fig. 2
[91]Open in a new tab
Venn diagram demonstrating the number of DEGs in each tissue type and
degree of overlapping in expression of those genes among the male (A)
and female (B) reproductive tissues. Male tissue samples include Tg:
the germinal zone of the testis; Tv: testis, part of vas deferens; Sv:
seminal vesicle. Female tissue samples include Og: the germinal zone of
the ovary; Ov: ovary, part of oviduct; Ut: uterus. The complete list of
DEGs is shown in Additional file [92]7: Table S4
Tissue-specific gene expression patterns
To identify genes preferentially transcribed in specific tissues,
hierarchical clustering was performed using the FPKM values of all A.
lumbricoides protein-coding genes expressed in the eight tissue samples
analyzed. The hierarchical clustering result revealed that 10,157 DEGs
were identified and classified into seven clusters (A–G) (Fig. [93]3),
of which DEGs were most classified in Cluster G (2935 DEGs), followed
by Clusters B (1988 DEGs), F (1844 DEGs), C (1199 DEGs), A (800 DEGs),
E (792 DEGs) and D (599 DEGs). Clusters C, D and E demonstrated unique
tissue specificity and were specifically expressed in Tg, Sv and Ut,
respectively, whereas Clusters A, B, F and G were predominantly
expressed in more than one tissue and were observed in Stf/Stm, Tv/Tg,
Og/Ov and Ov/Og, respectively. Based on the numbers of DEGs distributed
in specific tissue, > 50% of the total DEGs were predominantly
expressed in female reproductive tissue samples (5571 genes, 54.85%),
followed by the male reproductive tissue samples (3786 genes, 37.27%)
and male and female somatic tissue samples (800 genes, 7.88%) (see
Additional file [94]8: Table S5 for all genes expressed in at least one
tissue).
Fig. 3.
[95]Fig. 3
[96]Open in a new tab
Hierarchical clustering of Ascaris lumbricoides transcriptome profiles
to discrete tissue samples. Male tissue samples include Tg: the
germinal zone of the testis; Tv: testis, part of vas deferens; Sv:
seminal vesicle; Stm: male somatic tissue. Female tissue samples
include Og: the germinal zone of the ovary; Ov: ovary, part of oviduct;
Ut: uterus; Stf: female somatic tissue. Data from three biological
replicates were combined prior to clustering. Only a few DEG examples
were presented in the right box, which revealed DEGs preferentially
expressed in particular tissues. Tissue-specific clusters are labeled
A–G on the left and were used for subsequent tissue-specific functional
analysis. The number indicates the normalized expression and ranges
from white (no expression) to red (extremely high
expression). Additional file [97]8: Table S5 shows a list of all genes,
but only those that are expressed in at least one tissue
DEGs that are predominantly expressed in Stm and Stf tissue samples (Cluster
A)
Cluster A yielded results that were specific to Stm and Stf tissue
samples. It was expected that expressed genes associated with oxidative
phosphorylation would be predominant in somatic tissue. These included
acetylCoA_hyd_C domain-containing protein, cytochrome b-c1 complex
subunit, cytochrome b561 domain-containing protein, cytochrome c
domain-containing protein, cytochrome c oxidase subunit 1, cytochrome c
oxidase subunit 3 and succinate dehydrogenase [ubiquinone] flavoprotein
subunit and indicated that energy production was the primary activity
in this tissue. Additionally, several genes encoding cytoskeleton
structural components (Fig. [98]3 and Additional file [99]8: Table S5)
were also dominantly expressed in this tissue. This was expected since
the cuticle and musculature are major structural components of the
somatic tissue. These findings align with the active movement of the
worm in the intestine to protect itself from the weep-and-sweep
response in the expulsion mechanism [[100]37]. In addition to tubulin,
the target for benzimidazole drugs, other candidates involved in
movement and energy generation should be explored in the future for
innovative anthelminthic chemotherapy.
DEGs that are predominantly expressed in Tv and Tg tissue samples (Cluster B)
Cluster B was highly expressed in Tv and Tg tissue samples but was more
predominant in Tv. The enriched genes are associated with protein
phosphorylation, including protein kinase domain-containing protein,
serine/threonine-protein phosphatase and tyrosine-protein kinase, all
of which encode protein kinases which are pivotal regulators of
cellular function [[101]38]. When activated, these signaling enzymes
phosphorylate transcription factors and other intracellular proteins,
leading to alteration in gene expression or other cellular behavior and
activities [[102]38]. Moreover, protein kinases can drive multiple
important functions in addition to influencing one another through
cross-talk [[103]39]. The role of a serine/threonine kinase member,
vaccinia-related kinase, in Caenorhabditis elegans has been
investigated where it was found to be localized in the germline and
early embryos. Mutation of C. elegans vaccinia-related kinase resulted
in defects in germline, uterine and vulval development [[104]40,
[105]41]. Consistent with our finding in A. lumbricoides, the genes
encoding serine/threonine kinase and phosphatase were overrepresented
among the sperm-enriched genes in C. elegans, which suggests an
important function during spermatogenesis [[106]2]. In Schistosoma,
inhibitions of several protein kinases, e.g., Syk kinase, Src kinase
and polo-like kinases, impaired spermatogenesis and oogenesis, which
have also been proposed as a potential target for novel therapy
[[107]38, [108]42–[109]44]. Several genes encoding serine/threonine and
tyrosine kinases were found in spermatogenesis-enriched genes in a
study on A. suum germline transcriptomes [[110]2], which aligns with
data presented here. These potential targets in A. suum and A.
lumbricoides should be immediately selected and thoroughly
characterized for the development of anthelminthic drugs. Aside from
protein kinases, the genes encoding major sperm protein (MSP)
(Fig. [111]3 and Additional file [112]8: Table S5) were highly
expressed in this cluster; these are structural molecules that have
been reported to play a role in the motility of amoeboid sperm by the
regulated assembly and disassembly of MSP [[113]45, [114]46].
DEGs that are predominantly expressed in Tg tissue sample (Cluster C)
Genes in Cluster C were specifically expressed in Tg tissue, which are
involved in the cell-division cycle, including MCM_N domain-containing
protein, DNA replication licensing factor MCM7, histone H2A, histone
H2B, histone H3, histone H4 and dynein light chain and myosin_tail_1
domain-containing protein (Fig. [115]3 and Additional file [116]8:
Table S5). The strong expression of these genes might be due to
pronuclear migration and spindle assembly in the cell division cycle.
Furthermore, a set of genes associated with transcription, namely,
DNA-directed RNA polymerases, RPOLA_N domain-containing protein, F-box
domain-containing protein, cleavage and polyadenylation specificity
factor subunit 2, U2 snRNP auxiliary factor large subunit,
transcription initiation factor IIE subunit beta and C2H2-type
domain-containing protein, and a set of genes associated with
translation, namely eIF-5a domain-containing protein, translation
factor GUF1 homolog and several isoforms of ribosome domain-containing
protein, were predominantly expressed. The overexpression of these
genes suggests that protein synthesis is particularly active in this
tissue. Furthermore, other genes of interest, including several
isoforms of proteasome (proteasome endopeptidase complex, proteasome
subunit alpha and proteasome subunit beta as well as
ubiquitin_conjugat_2 domain-containing protein and E3 ubiquitin-protein
ligase) were also highly expressed in Tg. The ubiquitin-proteasome
system (UPS) is one of the most important proteolytic systems for
protein degradation and is associated with numerous dynamic cellular
processes [[117]47]. UPS maintains protein homeostasis in the male
reproductive system to regulate the progression of spermatogenesis at
various levels [[118]48]. Proteasome and ubiquitin have been proposed
as embryonic lethal genes in C. elegans because of the demonstration
that their inhibition causes cell division arrest to the embryo
[[119]49]. Inhibition of proteasomes has been reported to shorten the
longevity of C. elegans [[120]50]. In this regard, UPS may also be
essential for spermatogenesis and Ascaris viability.
DEGs that are predominantly expressed in Sv tissue sample (Cluster D)
Several genes associated with transmembrane transporter activity were
highly expressed in Sv tissue (Cluster D). Moreover, the group of genes
with cell adhesion function, including C-type lectin domain-containing
protein, CA domain-containing protein (cadherin), galectin and VWFA
domain-containing protein, was also highly expressed in Sv tissue.
Adhesion molecules play a crucial role in the early reproduction event,
including gamete transport, fertilization, embryonic development and
implantation [[121]51]. The function of these genes is predominantly
required for gamete fusion. As expected, the high expression of
adhesion molecules in this tissue may indicate that sperm are ready for
fertilization [[122]52]. In addition to Sv tissue, adhesion molecules
and metalloproteases (zinc metalloproteinases) were also detected in Ut
tissue to again facilitate gamete fusion. High expressions of
calponin-homology domain-containing protein, profilin and tubulin alpha
chain were also observed in Sv, indicating that the cytoskeleton
supports amoeboid sperm movement [[123]46].
DEGs that are predominantly expressed in Ut tissue sample (Cluster E)
Interrogation of Cluster E revealed that genes predominantly expressed
in Ut tissue were primarily focused on eggshell formation, including
chitin-binding type-2 domain-containing protein, col_cuticle_N
domain-containing protein and elongation of very long chain fatty acids
protein (Fig. [124]3 and Additional file [125]8: Table S5). While
embryo development may be supported by the expression of the genes
including cnd1 domain-containing protein, mothers against
decapentaplegic homolog, SPOC domain-containing protein and SUEL-type
lectin domain-containing protein. Interestingly, mothers against
decapentaplegic (Mad) homolog was discovered in this study. Mad is a
vital gene in Drosophila melanogaster, which contributes to the
development of the early embryo and 15 imaginal discs. Its mutation can
cause embryonic dorsal-ventral patterns and adult appendage defects
[[126]53–[127]55]. The Mad homolog, named SMA gene, was discovered in
C. elegans, and its alteration affects body size and male tail
development. The SMA gene encodes the signaling components of the
transforming growth factor-beta (TGF-β) signaling pathway, such as
sma-2, sma-3 and sma-4. The TGF-β signaling pathway contributes to body
size and dauer formation in C. elegans and is involved in innate
immunity, mesoderm and ectoderm patterning, longevity and fat
metabolism [[128]56]. Additionally, it was discovered that a component
of the TGF-β signaling pathway is localized in reproductive tissue
samples [[129]57] and plays a role in embryogenesis in Haemonchus
contortus [[130]58]. The expression of a signaling component in this
tissue was associated with the TGF signaling pathway, implying that
this route is crucial in Ascaris embryogenesis.
Several isoforms of genes encoding peptidases, including pepsin-I3
domain-containing protein, pepsin-I3 domain-containing protein,
peptidase S72 domain-containing protein, peptidase_M13
domain-containing protein and peptidase_M28 domain-containing protein,
were expressed in Ut tissue. It has been reported in C. elegans that
peptidases are actively expressed during embryogenesis and at the first
stage of larval development because of tissue remodeling and the
degradation of the nematode cuticle [[131]59]. Since peptidases have
been proposed as therapeutic and vaccine targets, our discovery
provides a novel target that potentially contributes to embryogenesis
and could be employed as a future target for effective control.
DEGs that are predominantly expressed in Og and Ov tissue samples (Cluster F)
In Cluster F (Og and Ov-specific), genes associated with the
cell-division cycle, including helicase, histone H2A, histone H3, DNA
replication licensing factor MCM7, DNA polymerase, Rad51
domain-containing protein, RECA_2 domain-containing protein and SMC_N
domain-containing protein, were predominantly expressed in Og tissue.
Furthermore, upregulation of genes involved in transcription and
translation was observed in this study (Fig. [132]3 and Additional file
[133]8: Table S5). In addition to the groups of genes mentioned above,
the Og tissue highly expressed several genes associated with protein
metabolism (Additional file [134]8: Table S5).
DEGs that are predominantly expressed in Ov and Og tissue samples (Cluster G)
Genes encoding receptors or activators associated with the
G-protein-coupled receptor (GPCR) signaling pathway and small
GTPase-mediated signal transduction pathway were discovered to be
expressed preferentially in Cluster G (Ov and Og specific), including
G_recep_F1_2 domain-containing protein, G-protein gamma
domain-containing protein, Rab-GAP TBC domain-containing protein and
Rho-GAP domain-containing protein. Both signaling pathways are
associated with cytoskeletal activity [[135]60, [136]61]. Rho-GAP (Rho
GTPase) domain-containing proteins act as molecular switches,
transducing signals by switching between an inactive and active
GTP-bound state. Signal transductions through these pathways regulate a
range of diverse cellular functions, including actin cytoskeleton
rearrangement, regulation of gene transcription, cell cycle regulation,
the control of apoptosis and membrane trafficking [[137]62, [138]63].
The Rho GTPase family has been reported to play a crucial role in
cytoskeleton organization, and our study discovered that a collection
of genes associated with cytoskeleton structure was expressed in Ov and
Og (Fig. [139]3 and Additional file [140]8: Table S5). The cellular
cytoskeleton undergoes significant changes during oogenesis due to cell
migration, membrane fusion and cytoskeleton remodeling [[141]64], and
our results showed that Rho GTPase may provide a vital function in
these tissues examined. Rho-1 inhibition in C. elegans caused early
embryonic arrest and cytokinesis failure, indicating the importance of
Rho signaling at the earliest stages of development [[142]65]. Rho
GTPase inactivation has been reported to cause embryonic mortality in
Drosophila and mice [[143]66]. In this regard, Rho GTPase may be a
promising target for Ascaris drug development.
Differential gene GO enrichment analysis
The enriched GO Terms of the male reproductive tissue samples
On the basis of the enriched GO Terms of the male reproductive tissue
samples, 52, 56 and 83 enriched GO Terms were identified for Tg, Tv and
Sv tissue samples, respectively, relative to the male somatic tissue.
Owing to the large number of enriched GO Terms, groupings were
performed under general terms (Fig. [144]4). Additional file [145]9:
Table S6 contains the complete list of enriched GO Terms for male
reproductive tissue. On the basis of DEGs under GO Terms, the top three
GO Terms for Tg tissue identified were protein phosphorylation (88
DEGs), phosphorylation (78 DEGs) and protein kinase activity (72 DEGs);
those for Tv tissue were ATP binding (224 DEGs), nucleotide binding
(141 DEGs) and protein phosphorylation (106 DEGs); those for Sv tissue
were an integral component of membrane (122 DEGs), membrane (83 DEGs)
and cytoplasm (61 DEGs). When analyzing the top GO Terms based upon
statistical significance (P-value), the three most significant GO Terms
for Tg were protein kinase activity, phosphoprotein phosphatase
activity and phosphorylation; those for Tv were protein
phosphorylation, protein kinase activity and protein serine/threonine
kinase activity; those for Sv were cytoskeleton, an integral component
of membrane and actin binding.
Fig. 4.
[146]Fig. 4
[147]Open in a new tab
GO Term significantly enriched (P-value < 0.05) in the male discrete
reproductive tissues. Red, blue and yellow boxes represent Tg (the
germinal zone of the testis), Tv (testis, part of vas deferens) and Sv
(seminal vesicle) tissues, respectively. Overlapping colored boxes
represent GO Terms enriched between tissue samples. Due to the large
number of GO Terms enriched within each tissue sample, groupings under
general terms were used. The complete list of tissue-enriched GO Terms
is shown in Additional file [148]9: Table S6
According to the Go Term findings, both Tg and Tv tissues presented a
set of genes related to protein phosphorylation, which included a range
of activities: kinase, phosphoprotein phosphatase, phosphorylation,
protein dephosphorylation, protein kinase activity, protein
phosphorylation and protein serine/threonine kinase activity. These
findings corroborated the DEG hierarchical clustering, which
highlighted that protein phosphorylation, especially kinase activity,
is significantly expressed in Tg and Tv tissue samples, which is likely
to be essential for spermatogenesis.
The majority of the GO Terms specific to Sv tissue were associated with
carbohydrate metabolism and respiratory chain reactions. Genes
associated with these functions included phosphogluconate dehydrogenase
activity, NADH dehydrogenase activity, GDP-mannose 4,6-dehydratase
activity, mannosidase activity, GDP-mannose metabolic process,
carbohydrate metabolic process, oxidation-reduction process and
respiratory chain, implying a requirement of strong energy output
promotes sperm movement within this tissue.
Based on the functional enrichment in the male reproductive tissues of
A. suum [[149]16], our findings were concordant in terms of high
phosphatase and kinase activity in the Tg and Tv tissues. The
enrichment of the GO term "phosphoprotein phosphatase activity"
(GO:0004721) supports the presence of high phosphatase activity, and
the enrichment of three different GO terms describing kinase activity,
such as "protein kinase activity" (GO:0004672), "kinase activity"
(GO:0016301) and "protein serine/threonine kinase activity"
(GO:0004674), supports the presence of high kinase activity. MSP was
also discovered to be overexpressed in the testis of A. suum. Our study
supported that MSP and MSP domain-containing proteins were both
substantially expressed in the testes. Our study, however, also
revealed that genes were more prominent in the Tg and Tv tissues.
Furthermore, our findings confirmed the presence of actin and
cytoskeleton activity in the previous study, which has been reported to
be crucial for nematode sperm motility and activation [[150]46]. This
was due to the enrichment of several different GO Terms that indicated
actin and cytoskeleton activity in the Sv tissue, including “actin
binding” (GO:0003779), “actin filament” (GO:0005884), “actin filament
binding” (GO:0051015), “barbed-end actin filament capping”
(GO:0051016), “cytoskeleton” (GO:0005856), “cytoskeleton organization”
(GO:0007010) and “motor activity” (GO:0003774).
The enriched GO Terms of the female reproductive tissue samples
A total of 50, 43 and 72 GO Terms were identified for Og, Ov and Ut
tissue samples, respectively, relative to the female somatic tissue.
The top three GO Terms for Og tissue were binding (66 DEGs), actin
binding (22 DEGs) and locomotion (22 DEGs); those for Ov tissue were
binding (66 DEGs), calcium ion binding (41 DEGs) and transmembrane
transport (35 DEGs); those for Ut tissue were metal ion binding (49
DEGs), structural constituent of ribosome (30 DEGs) and translation (30
DEGs) (Additional file [151]9: Table S6). When analyzing the GO Term
based on P-value, the top three most significant GO Terms for Og were
respiratory chain, cysteine-type peptidase activity and glutamine
metabolic process; those for Ov were cysteine-type peptidase activity,
cell cycle and glycolytic process; those for Ut were cytokinesis,
heterotrimeric G-protein complex and serine-type endopeptidase
inhibitor activity. Additional file [152]9: Table S6 contains the
complete list of enriched GO Terms for female reproductive tissue.
The high activity of cytokinesis indicative of embryo development
within Ut tissue was reflected in the enriched GO Terms (Fig. [153]5)
associated with structural components and cytoskeleton organization.
Genes within these GO Terms included barbed-end actin filament capping,
actin binding, microtubule binding, cytoskeleton, myosin filament and
myosin complex. Furthermore, heterotrimeric G-protein complex was the
second significantly enriched GO Term, indicating that genes associated
with G-protein complex, including adenylate cyclase-modulating GPCR
signaling pathway, G-protein beta/gamma-subunit complex binding and
GPCR signaling pathway, were predominantly expressed in Ut tissue. The
GO Term for GPCR activity was also found in both Og and Ov tissue
samples (Fig. [154]5). According to the results mentioned in the
previous section, the GPCR signaling pathway and small GTPase-mediated
signal transduction pathway may play a pivotal role in cytoskeleton
organization due to cytokinesis. Our finding highlights the roles of
the GPCR signaling pathway and small GTPase-mediated signal
transduction pathway in cytoskeleton organization [[155]60, [156]61,
[157]67] due to cytokinesis in oogenesis and embryogenesis of female
reproductive tissue.
Fig. 5.
[158]Fig. 5
[159]Open in a new tab
GO Term significantly enriched (P-value < 0.05) in the female discrete
reproductive tissues. Red, blue and yellow boxes represent GO Term
enriched in Og (the germinal zone of the ovary), Ov (ovary, part of
oviduct) and Ut (uterus), respectively. Overlapping colored boxes
represent GO Terms enriched between tissue samples. Due to the large
number of GO Terms enriched within each tissue sample, groupings under
general terms were used. The complete list of tissue-enriched GO Terms
is shown in Additional file [160]9: Table S6
Rosa et al. (2014) discovered that DNA binding and replication were
among the top five enriched GO terms in the ovary of A. suum [[161]16].
This result was consistent with our findings, which identified several
GO terms that related to these biological functions in the Og and Ov
tissues, including "nucleosome" (GO:0000786), "damaged DNA binding"
(GO:0003684) and "cell cycle" (GO:0007049). This result demonstrated
that our approach was successful in identifying the expected function
in the ovary. Moreover, our finding supported the presence of
phosphatidylinositol signaling in the ovary of A. suum as the PIPK
domain-containing protein associated with phosphatidylinositol
phosphate kinase activity (GO:0016307) was identified in the Ov and Og
tissues. Phosphatidylinositol signaling has been reported in the
previous study to be necessary for ovulation of in C. elegans
[[162]68]. The previous study reported that “protein binding”
(GO:0005515) and “catalytic activity” (GO:0003824) were associated with
the A. suum uterus. Our findings, however, showed that there were other
GO Terms of Ut tissue that indicated "protein binding" and "catalytic
activity," such as “protein domain specific binding” (GO:0019904),
“sulfuric ester hydrolase activity” (GO:0008484), “cysteine-type
peptidase activity” (GO:0008234), “GTP cyclohydrolase I activity”
(GO:0003934), “hydroxyacid-oxoacid transhydrogenase activity”
(GO:0047988) and “glutamine-fructose-6-phosphate transaminase activity”
(GO:0004360). This finding provided a comprehensive view of gene
expression in Ascaris reproductive tissues.
KEGG enrichment analysis
On the basis of the rich factor (Fig. [163]6A), the top three most
significant KEGG terms for Tg tissue were the Hippo signaling pathway –
fly (ko04391), phototransduction – fly (ko04745) and arrhythmogenic
right ventricular cardiomyopathy (ko05412). For Tv tissue, the top
three most significant KEGG terms include the ErbB signaling pathway
(ko04012), rheumatoid arthritis (ko05323) and non-small cell lung
cancer (ko05223), whereas the top three most significant KEGG terms for
Sv tissue were the rap1 signaling pathway (ko04015), oxytocin signaling
pathway (ko04921) and tight junction (ko04530) (see Additional file
[164]10: Table S7 for complete list of enriched KEGG for male
reproductive tissue). In female reproductive tissues, the top three
most significant KEGG terms for Og tissue were platelet activation
(ko04611), the oxytocin signaling pathway and the Hippo signaling
pathway-fly. Tight junction, apoptosis (ko04210) and the oxytocin
signaling pathway were the top three most significant KEGG terms
enriched in Ov tissue. The top three most significant KEGG terms for Ut
tissue were metabolic pathways (ko01100), tight junction and
shigellosis (ko05131) (Fig. [165]6B). The complete list of enriched
KEGG for female reproductive tissue is available in Additional file
[166]10: Table S7.
Fig. 6.
[167]Fig. 6
[168]Open in a new tab
Scatter plot of differential gene KEGG enrichment of male A and female
B reproductive tissues. RichFactor is on the X axis, while KEGG
pathways are presented on the Y axis. The number of DEGs in the pathway
is positively associated with the size of the dot. Distinct Q-value
ranges are indicated by different color codes
The results of the KEGG enrichment analysis indicated that the Hippo
signaling pathway-fly was common to both Tg and Og tissue samples,
suggesting that this pathway is predominantly specific to both male and
female germinal tissue samples. Most of the genes involved in this
pathway of Tg and Og tissue samples were similar, such as actin
beta/gamma 1 (ACTB_G1), transcriptional enhancer factor (TEAD), 14-3-3
protein epsilon (YWHAE), serine/threonine-protein phosphatase 2A
catalytic subunit (PPP2C), serine/threonine-protein phosphatase 2A
regulatory subunit B (PPP2R2) and serine/threonine-protein kinase
LATS1/2 (LATS1_2) (Additional file [169]11: Figure S4 and Additional
file [170]12: Table S8). The Hippo signaling pathway-fly is the
signaling pathway that promotes cell apoptosis and restricts organ size
overgrowth, indicating that this pathway may be involved in control
during the early phase of germinal cell division in oogenesis and
spermatogenesis. In addition, it has been reported in C. elegans that
the Hippo signaling pathway is essential for maintenance of apicobasal
polarity in the growing intestine [[171]69].
The oxytocin signaling pathway was indicative of both male (Sv tissue)
and female reproductive tissue samples (Og and Ov tissue samples)
(Additional file [172]11: Figure S4). In depth into the pathway, there
are six genes that these tissue samples shared in common, including
actin beta/gamma 1 (ACTB_G1), ryanodine receptor 2 (RYR2), Ras homolog
gene family member A (RHOA), calmodulin (CALM), inositol
1,4,5-triphosphate receptor type 1 (ITPR1) and adenylate cyclase 9
(ADCY9). Although eight genes are shared by the Og and Ov tissue
samples, but not the Sv tissue [atrial natriuretic peptide receptor A
(ANPRA), calcium/calmodulin-dependent protein kinase I (CAMK1), guanine
nucleotide-binding protein G(o) subunit alpha (GNAO), guanine
nucleotide-binding protein G(s) subunit alpha (GNAS), GTPase Kras
(KRAS), serine/threonine-protein phosphatase PP1 catalytic subunit
(PPP1C), serine/threonine-protein phosphatase 2B catalytic subunit
(PPP3C) and 5′-AMP-activated protein kinase, catalytic alpha subunit
(PRKAA)], this indicates that certain genes play a role in Og and Ov
tissue.
Furthermore, elongation factor 2 (EEF2), guanine nucleotide-binding
protein G(q) subunit alpha (GNAQ) and classical protein kinase C alpha
type (PRKCA) were shown to be specific for Og, Ov and Sv, respectively.
Additional file [173]12: Table S8 contains the complete list of
significant DEGs annotated in KEGG enrichment.
Although oxytocin neuropeptides are strongly implicated in mammalian
reproductive and social behaviors, an oxytocin/vasopressin-like
signaling pathway was identified in sexually dimorphic patterns of C.
elegans. This pathway involves a peptide, nematocin (encoded by ntc-1)
and two receptors (encoded by ntr-1 and ntr-2), which are associated
with the GPCR superfamily [[174]70]. Mutations in the ntc-1 gene or its
receptors cause defects in reproductive behavior, such as mate
searching, mate recognition and mating [[175]71]. As a result, the
properties and functions of the signaling molecules involved in the
oxytocin signaling pathway in Ascaris should be further investigated to
identify prospective therapeutic targets.
Tight junction was another crucial pathway discovered in both male and
female reproductive tissue samples (Additional file [176]11: Figure
S4). Several genes could be commonly identified in Ov, Ut and Sv tissue
samples, including actin beta/gamma 1 (ACTB_G1), integrin beta 1
(ITGB1, CD29), myosin heavy chain (MYH), E3 ubiquitin-protein ligase
NEDD4-like (NEDD4L), Ras-related protein Rab-8A (RAB8A, MEL) and
tubulin alpha. Additionally, myosin regulatory light chain 2 (MYL2),
Ras-related C3 botulinum toxin substrate 1 (RAC1) and Ras homolog gene
family, member A (RHOA) were discovered in Ov and Sv tissue samples but
not in Ut tissue. Although serine/threonine-protein phosphatase 2A
catalytic subunit (PPP2C), 5′-AMP-activated protein kinase, catalytic
alpha subunit (PRKAA, AMPK), atypical protein kinase C iota type
(PRKCI) and Ras-related protein Rap-1A (RAP1A) were specifically
discovered in Ov tissue, this suggests that those genes play a unique
role in Ov tissue (Additional file [177]12: Table S8). According to
pathway mapping, genes involved in this pathway are associated with
actin assembly, adherens junction assembly, tight assembly, cell
polarity and cell migration, implying that chromosome organization is
active because of cell division in reproductive tissue.
Validation of gene expression by RT-qPCR
Seven tissue-specific genes were selected as a representative of each
cluster. All selected tissue-specific genes demonstrated consistent
expression, as revealed in the hierarchical clustering analysis
(Additional file [178]13: Figure S5). ALUE_0000279901, ALUE_0002034701,
ALUE_0001490701, ALUE_0000300701, ALUE_0000531301, ALUE_0000064301 and
ALUE_0001851601 genes were expressed specifically to Tg, Sv, Ut,
Stf/Stm, Tv/Tg, Og/Ov and Ov/Og tissue samples, respectively. On the
basis of our transcriptome data, RT-qPCR was performed in this study
using two putative housekeeping genes, namely, eIF6 and NADH cytochrome
b5 reductase genes. However, only the eIF6 gene was discovered to
display stable gene expression in all reproductive tissue samples and
could be used to normalize target gene expression.
Conclusion
This study generated transcriptome profiles of discrete reproductive
and somatic tissue samples from male and female A. lumbricoides. On the
basis of the FPKM profiles, gene expression analysis was used to
identify DEGs from each distinct tissue type examined. Hierarchical
clustering analysis identified seven clusters associated with specific
tissues and groups of tissues. The findings revealed that DEGs involved
in protein phosphorylation were differentially expressed particularly
in Tv and Tg tissue samples and played a crucial role in
spermatogenesis. Although DEGs were shared between Tg and Tv tissue
samples, adhesion molecules were specifically identified in Sv tissue
and played a critical role in the fertilization process. Several DEGs
associated with the cell division cycle and transcription process were
identified in Og and Ov tissue samples. DEGs involved in GPCR signaling
pathway and small GTPase-mediated signal transduction pathway were
discovered to play a crucial role in cytoskeleton organization due to
oogenesis in Ov and Og tissue samples. DEGs associated with the SMA
genes and TGF-β signaling pathway were discovered to be crucial in the
embryogenesis of Ut tissue. Additionally, Hippo signaling, oxytocin
signaling and tight junction pathways were identified to play a role in
Ascaris male and female reproductive systems. Some of the genes
discovered in this study that are related to protein phosphorylation,
the TGF-signaling system and the oxytocin signaling pathway have been
linked to defects in germline development, embryogenesis and
reproductive behavior. In addition to providing the transcriptome
profiles of discrete reproductive tissue samples of A. lumbricoides,
the results of this study highlight genes that are likely to play
significant roles in the functions of these important tissues. Data
generated here will inform appropriate selection of gene targets for
therapeutic exploitation to aid future sustained control of ascariasis.
Supplementary Information
[179]13071_2022_5602_MOESM1_ESM.pptx^ (2.2MB, pptx)
Additional file 1: Figure S1. The dissection of somatic and
reproductive tissues.
[180]13071_2022_5602_MOESM2_ESM.docx^ (19.3KB, docx)
Additional file 2: Table S1. The primer list for constructing library
and validating gene expression.
[181]13071_2022_5602_MOESM3_ESM.docx^ (26KB, docx)
Additional file 3: Table S2. Sequencing data and quality assessment of
A. lumbricoides discrete tissue samples.
[182]13071_2022_5602_MOESM4_ESM.xlsx^ (17.6KB, xlsx)
Additional file 4: Table S3. Raw data, filtered data, and data
alignment statistics.
[183]13071_2022_5602_MOESM5_ESM.pptx^ (1.7MB, pptx)
Additional file 5: Figure S2. The distribution of reads in different
genomic regions of somatic and reproductive tissue samples.
[184]13071_2022_5602_MOESM6_ESM.pptx^ (6.1MB, pptx)
Additional file 6: Figure S3. Overall quality assessment of RNA
sequencing.
[185]13071_2022_5602_MOESM7_ESM.xlsx^ (8MB, xlsx)
Additional file 7: Table S4. The complete list of significant DEGs
comparing between the tissue samples.
[186]13071_2022_5602_MOESM8_ESM.xlsx^ (3.4MB, xlsx)
Additional file 8: Table S5. List of all genes for hierarchical
clustering of DEGs.
[187]13071_2022_5602_MOESM9_ESM.xlsx^ (52.2KB, xlsx)
Additional file 9: Table S6. The complete list of GO Terms
significantly enriched in A. lumbricoides discrete reproductive
tissues.
[188]13071_2022_5602_MOESM10_ESM.xlsx^ (126.7KB, xlsx)
Additional file 10: Table S7. The complete list of KEGG pathway
significantly enriched in A. lumbricoides discrete reproductive
tissues.
[189]13071_2022_5602_MOESM11_ESM.pptx^ (1.5MB, pptx)
Additional file 11: Figure S4. Demonstration of important signaling
pathways, including the Hippo signaling pathway-fly, Oxytocin signaling
pathway and tight junction pathway
[190]13071_2022_5602_MOESM12_ESM.xlsx^ (55.9KB, xlsx)
Additional file 12: Table S8. The complete list of significant DEGs
annotated in KEGG enrichment.
[191]13071_2022_5602_MOESM13_ESM.pptx^ (307.7KB, pptx)
Additional file 13: Figure S5. Gene expression level of tissue-specific
genes in A. lumbricoides discrete tissues
Acknowledgements