Results
Blocking primers
development
We discovered three unique regions in M. festivus rRNA 18S V4-V5
gene consensus sequence following its comparison with seven potential
fish parasites [Fig. 1]. One region was located 5’ of the consensus
sequence and had a length of 24 pb, a second one was located at the
middle of the sequence and had a length of 40 pb, and the third was
located 3’and had a length of 28 pb. From these regions, two
combinations of blocking primers were developed, the F-primers[635F-C3 and 1062R-C3] and the M-primers [816F-C3 and
846R-C3], respectively located at the extremities and at the middle of
the target gene.
Blocking primer
evaluation
The relative abundance of host DNA in samples amplified using theF-Primers was not significantly different from the control group
[Fig. 2]. Indeed, none of the three concentrations ofF-primers significantly reduced the number of host amplicons in
samples according to the paired samples Wilcoxon test (p-values
> 0.05). Also, using an increasing concentration ofF-primers , from 2X to 10X, did not improve its effectiveness and
led to inconsistent results in samples with a low host DNA to gut
content ratio [Table 1].
According to the paired sample Wilcoxon test, the M-primers at a
concentration of 2X allowed a significant reduction of the mean relative
abundance of host sequences present in samples when compared to the
control group (p-value = 0.004). The mean relative abundance of the
class Actinopterygii, the taxonomic class of M. festivus , in theM-primers group represented 34.01 % of the abundance of the
control group [Fig. 2]. Also, the M-Primers efficacy at inhibiting
host’s DNA amplification was significative in samples starting with a
low and a high host DNA to gut content ratio [Table 1]. For example,
the mean relative abundance of the class Actinopterygii in gut IDs 1 to
4 of the M-primers group represents 14.58 % of the relative
abundance of the control group. Furthermore, host DNA relative abundance
in gut IDs 7 and 8 respectively reduced from 72 and 77.9 % to 24 and
21.5 %. However, using a concentration of more than 2X of theM-primers completely inhibited the PCR amplification [Table
1].
Alpha diversity
The Faith phylogenetic alpha diversity index was significantly lower in
the M-Primers group than in the control group in the Tukey HSD
test (p-value = 0.02) [Fig. S2]. However, the total number of ASVs
(observed diversity), the Shannon and the Simpson alpha diversity
indexes were not significantly lower when comparing the same groups
(p-value > 0.05). Furthermore, when omitting the class
Actinopterygii, there was no significant difference in alpha diversity
between the M-primers group and the control group in the Tukey
HSD test (p-values > 0.05) [Fig. S3]. For theF-Primers , their effect was inconsistent at different
concentrations and did not significantly influence the alpha diversity
in samples. Overall, we observed a mean of 30 ASVs per sample, with
20 % of these from the Phylum Vertebrata. Also, two genera of plants,Cyperus sp. and Oryza sp. , were the most abundant ASVs for
the samples analysed. These two genera, from the order Poales, averaged
48 % of the total relative abundance in samples.
Beta diversity
According to the two-way PERMANOVA comparing Bray-Curtis dissimilarity
indexes and considering the eight gut IDs and the four blocking primer
combinations as factors, most of the variance was explained by
differences between gut IDs (R2 = 0.85; p-value = 3.0
x 10-5) and a moderate part of the variance was
explained by differences between blocking primer groups
(R2 = 0.07; p-value = 1.0 x 10-4).
Furthermore, the M-primers was by far the combination of blocking
primers with the most influence on the sample’s beta diversity in the
Pairwise Adonis test (For M-primers , R2 = 0.09;
p-value = 0.007).
In a PCoA based on Bray-Curtis’s dissimilarity indexes, there was a
significant shift in beta diversity in the four samples which contained
a high ratio of host DNA to gut content (Gut IDs: 5 to 8), but no
important variation in beta diversity for the four gut samples with a
low abundance of host DNA contamination (Gut IDs: 1 to 4) [Fig. 3].
This result was very similar when producing a PCoA based on weighted
Unifrac distances [Fig. S4]. For samples 5 to 8, the usage ofM-primers led to a shift in beta diversity, reducing the
dissimilarity between gut IDs 5 to 8 and 1 to 4 [Fig. 3].
Consequently, when computing a pairwise Adonis test using a dataset
omitting the class Actinopterygii, M-Primers did not have a
significant effect on the beta diversity of samples when compared to the
control group (R2 = 0.03; p-value = 0.07). Again, theF-Primers had little influence on the beta-diversity in samples
and showed inconsistent patterns that did not support their
effectiveness at blocking host DNA amplification.
Blocking primers for parasite
detection
The usage of M. primers enhanced the detection of low abundance
parasitic taxonomic classes in gut IDs 5 to 8 [Fig. 4]. For
instance, we detected 12 different taxonomic classes in theM-primers group against 9 in the group without blocking primers
in the top 10% ASVs barplots [Fig. 4]. From these, M-Primersenhanced the detectability of Trematode in gut ID 6 and Arcellinida in
gut ID 7. Also, these classes were detected in higher relative abundance
in the M-Primers group. Also, a parasitic Ciliophora from the
genus Nyctotherus sp. was detected in high relative abundance in
two gut samples (Gut IDs 2 and 7) and in lower abundance in one (Gut ID
4) [Fig. 4]. Finally, we detected the presence of a Tubilinea in gut
ID 7 and a very low relative abundance of microsporidia in gut ID 4.