# Expression pattern of genes involved in biomineralization in black and orange mantle tissues of pearl oyster, Pinctada persica

Fatemeh Parvizi, Arash Akbarzadeh, Ahmad Farhadi, Sophie Arnaud-Haond, Mohammad Sharif Ranjbar

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Fatemeh Parvizi, Arash Akbarzadeh, Ahmad Farhadi, Sophie Arnaud-Haond, Mohammad Sharif Ranjbar. Expression pattern of genes involved in biomineralization in black and orange mantle tissues of pearl oyster, Pinctada persica. Frontiers in Marine Science, 2023, 9, 10.3389/fmars.2022.1038692. hal-04032806

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EDITED BY

Hyun Woo Kim,
Pukyong National University,
Republic of Korea

REVIEWED BY

Dahui Yu,
Beibu Gulf University, China
Huayang Guo,
Chinese Academy of Fishery Sciences
(CAFS), China

*CORRESPONDENCE

Mohammad Sharif Ranjbar

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sharif.ranjbar@hormozgan.ac.ir

Arash Akbarzadeh

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akbarzadeh@hormozgan.ac.ir

SPECIALTY SECTION

This article was submitted to
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Bioproducts,
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Frontiers in Marine Science

RECEIVED 07 September 2022

ACCEPTED 23 December 2022

PUBLISHED 19 January 2023

CITATION

Parvizi F, Akbarzadeh A, Farhadi A, Arnaud-Haond S and Ranjbar MS (2023) Expression pattern of genes involved in biomineralization in black and orange mantle tissues of pearl oyster, *Pinctada persica*. *Front. Mar. Sci.* 9:1038692.
doi: 10.3389/fmars.2022.1038692

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© 2023 Parvizi, Akbarzadeh, Farhadi, Arnaud-Haond and Ranjbar. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Expression pattern of genes involved in biomineralization in black and orange mantle tissues of pearl oyster, *Pinctada persica*

Fatemeh Parvizi¹, Arash Akbarzadeh¹*, Ahmad Farhadi², Sophie Arnaud-Haond³ and Mohammad Sharif Ranjbar⁴*

¹Department of Fisheries, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, Iran, ²Department of Natural Resources and Environmental Engineering, School of Agriculture, Shiraz University, Shiraz, Iran, ³Marine Biodiversity Exploitation and Conservation (MARBEC), UMR 9190, Univ. Montpellier, IFREMER, IRD, CNRS, Sète, France, ⁴Department of Marine Biology, Faculty of Marine Science and technology, University of Hormozgan, Bandar Abbas, Iran

A few species of mollusks display color variation in their soft tissues. In pearl oysters, the color polymorphism in mantle tissue is associated with the color and radiance of shell and pearl. The study of biomineralization related genes in mantle tissue of pearl oysters can be used as a suitable approach to better identify the molecular mechanisms that influence shell and pearl quality and color variations. In this study, we investigated the expression of biomineralization-related genes in black and orange mantle morphotypes of pearl oyster, *Pinctada persica* in both warm and cool seasons using quantitative real-time PCR. Our results showed that the genes involved in biomineralization of the prismatic and nacre layer, i.e.; ASP, KRMP, MRNP34, SHELL, SHEM1B, LINKINE, PIF, SHEM5, NACREIN, and in pigmentation (TYR2A) were significantly higher expressed in orange phenotype compared to those of black one. The higher expression of ASP, KRMP, SHEM5, LINKINE and NACREIN in orange phenotype was only observed in warm season, but PIF, SHELL, SHEM1B, and TYR2A were upregulated in both warm and cool seasons. These results suggest the existence of different genetic processes between the two color morphs of *P. persica* and the more active role of genes in orange morphotype, particularly in warmer season. This study provides better understanding of the molecular mechanisms underlying biomineralization in pearl oysters.

KEYWORDS

gene expression, mantle color, biomineralization, qRT-PCR, pearl oyster
Introduction

Pearl oysters occur around the world with considerable
variation in the shell and pearl phenotype in each species
(Strack, 2008; Fougerouse et al., 2008). Although the shell color
variation in intra and inter species of mollusks is common, color
variation in soft tissues has been reported for only few species (Ky
et al., 2019). For example, the black-lip pearl oyster (Pinctada
margaritifera), a widely distributed Indo-Pacific pearl oyster
species (Strack, 2008), consists of three distinct phenotypic color
morphs of mantle tissue i.e., black, albino, and orange (Shirai and
Nakamura, 1994; Hwang and Okutani, 2003; Wada and Tëmkin,
2008). However, the individuals with black mantle phenotype are
the most frequent, and the albino and orange morphotypes are
rare mutants (Ky et al., 2019). On the contrary, P. persica which is
the most important pearl oyster species of Persian Gulf, displays
two mantle color morphotypes, orange and black, but the black
phenotype is rare and the orange morphotype is found at higher
frequencies (Parvizi et al., 2017). P. persica was previously known
as P. margaritifera var. persica (Jameson, 1901; Lal et al., 2017),
but recently has been recognized as a new species (Ranjbar et al.,
2015). The cross examination on P. margaritifera demonstrated
that the variations in mantle color follow the Mendelian
inheritance pattern (Ky et al., 2019). Ky et al. (2016) showed
that the orange allele of P. margaritifera is recessive to the black
one (Ky et al., 2016), and therefore the black mantle color is the
dominant morphotype of the species. It is likely that the black
phenotype of P. persica is the mutant phenotype, but it should
receive more research attention.

Studies have shown that in pearl oysters, the color
polymorphism of mantle tissue is linked to the color of shell
and pearl (Ky et al., 2019) dependent of transcriptional activity of
biomineralization genes leading to shell and pearl formation
(Wada, 1983; Clark et al., 2010; Joubert et al., 2010; Gardner
et al., 2011; Shi et al., 2013; Freer et al., 2014; Sun et al., 2015;
Wang et al., 2017). The color variation of shell is result of genes
involved in different functions from shell structure to pigment
synthesis. Differential expression of many genes known to be
involved in the formation of the calcitic layer (e.g Aspein,
Prismalin, Shematin and etc), suggesting that variation in shell
color could primarily take place in the calcitic layer (Lemer et al.,
2015). Moreover, the aragonite binding pigments related to the
biomineralization process also play a role in the color of the
nacreous layer (Snow et al., 2004). Difference in expression
patterns of some genes (like Pif-177, Collagen alpha-1 (XI)
chain precursor, Zinc metalloprotease), involved in the
formation of the aragonite tablets of the nacre and in the
biosynthesis chain of melanin, suggesting that color
polymorphism occur at different levels in the shell structure
(Lemer et al., 2015). Genes involved in the melanin pathway
including tyrosinase which is known as a key biomineralization
enzyme (Sun et al., 2015) are among the other potential elements
of the shell layers coloration process (Lemer et al., 2015).

The study of biomineralization related genes expression in
mantle tissue can be used as a suitable approach to better identify
the differentially expressed genes in different morphotypes and
their potential role in shell and pearl quality and color variations.
Lemer et al. (2015) and Ky et al. (2019) investigated the expression
of some candidate genes associated to different mantle
morphotypes in *P. margaritifera*. Using suppressive and
subtractive hybridization (SSH) method and RT-qPCR, Lemer
et al. (2015) identified differentially expressed genes between black
and full albino phenotypes of *P. margaritifera* (Lemer et al., 2015).
Comparing the differential expression of a set of genes in black
*versus* orange mutant phenotypes, both in graft tissue and pearl
sac, Ky et al. (2019) also underlined the existence of differential
expression of four upon six genes and underlined very different
pathways in the orange phenotypes than in the albino ones. Their
study also revealed a strong influence of the orange phenotype on
the color of pearl, leading to lighter colors that may be an
interesting source of diversification for jewellery (Ky et al.,
2019). The black phenotype shell has dark color and the orange
one exhibit red outer shell phenotype (Ky et al., 2016). The inner
shell of orange phenotype, in the contact area with the mantle at
the edge of the shell (band color), is paler than that of the black
type (Ky et al., 2019).

The pearl oyster *P. persica* inhabiting the shallow and warm
waters of the Persian Gulf are known to produce the most
famous pearl qualities in the world. The pearl production in this
region and in this species is believed to be one of the first of its
kind in the world (Bowen, 1951). Unlike its closely related
species (*P. margaritifera*), *P. persica* has a distinct phenotypic
color morph of mantle tissue, so that the orange mantle color of
*P. persica* is the dominant morphotype compared to the
black one.

Altogether the unique morphological characteristic,
economic value and decreasing trend of population, this
species could also be considered as an interesting pearl oyster
species to investigate the molecular mechanisms involved in
phenotypic variation, particularly for color traits, and
biomineralization processes of shell and pearl formation.
Using a deep literature mining across published studies in
pearl oysters, we selected a suite of genes involved in
biomineralization, shell formation and color polymorphism
in pearl oysters, and then evaluated their expression in the
mantle tissue of two morphotypes of *P. persica* using
quantitative reverse transcription PCR (qRT-PCR). In
molluscan, temperature is one of the important factors
influencing physiological processes (Taylor and Venn, 1979;
Park, 2001) and a marked effect has been reported on
biomineralization (Yao and Somero, 2014). Given the
potential influence of the environmental factors, i.e.,
temperature on transcriptome and biomineralization (Yao
and Somero, 2014; Li et al., 2016), we also investigated the
expression of biomineralization genes in black and orange
morphotypes of *P. persica* in both warm and cool seasons.
Materials and methods

Thirty-one adult species of *P. persica* from two different
color phenotypes of mantle tissue (21 orange and 10 black;
Figures 1A, B) with 100–170 mm in dorso-ventral length were
collected from Larak Island, Persian Gulf (26° 53.385′ N. 56 °
21.061′ E; Figure 1C) by scuba diving during warm (September
2019, 32 °C) and cool (March 2020, 23 °C) seasons. The sample
size including six black and 12 orange morphotypes collected in
warm season, plus four black and nine orange phenotypes

sampled in cool season. The graft part of the mantle tissue was
dissected and stored at –80°C until subsequent RNA extraction.

RNA was extracted from 50 mg of the mantle tissue using
Trizol® reagent (GeneAll, Korea) in RNase free environment,
according to the manufacturer's recommendations with some
modifications to improve the purity of RNAs. The tissues were
homogenized in liquid nitrogen and treated in Trizol® reagent,
accordingly. RNA was precipitated in isopropanol and washed in
75% ethanol two times and finally eluted in 50 ul DEPC treated
water (Biobasic, Canada). RNA was quantified using a

<figure><img src="image_5.png" /></figure>
Nanodrop spectrophotometer (Thermo Scientific, MA) and the quality of the RNA was checked for degradation using 1% (w/v) agarose gel electrophoresis. Total RNA for each individual was then treated with DNase reagent (Thermo Scientific, MA) to cleanup potential DNA residuals. Afterwards, cDNA was synthesized using 1µg of total RNA as a template, and a cDNA synthesis kit (SMOBio, Taiwan) containing 1 µl dT primer and 0.2 µl random primers, following the manufacturer's instructions.

In order to identify the relative gene expression profiles of black and orange mantle tissues, 16 candidate genes were targeted based on their functions (Table 1). The selected genes were divided into 5 categories (defined based on Lemer et al. (2015) and Ky et al. (2019) studies) as follows; 1] genes involved in the formation of the prismatic layer: Aspein, KRMP, Peroxidas, Shematrin1, Prismalin, Shematrin5 and Calmodulin (Suzuki et al., 2004; Tsukamoto et al., 2004; Yano et al., 2006; Zhang et al., 2006; Marie et al., 2012; Lemer et al., 2015; Le Luyer

<table><caption>TABLE 1 Details of the candidate genes and the primer pairs used for gene expression analysis in *Pinctada persica* in this study.</caption><thead><tr><th>Gene name</th><th>Gene description</th><th>Function</th><th>Seq (5'-3')</th><th>Amplicon Length (bp)</th><th>Accession number and citation</th></tr></thead><tbody><tr><td>ASP<sup>1</sup></td><td>Aspein shell matrix protein</td><td>Prismatic formation</td><td>TCACAATGAAGGGGATAGCC<br>TACGCCTGATGACCCTAGTT</td><td>110</td><td>AB685319.1</td></tr><tr><td>CALM</td><td>Calmodulin</td><td>Prismatic formation</td><td>ATAACAACCAAGGAGCTGGGT<br>TTCCGTTTCCATCAGCGTCTA</td><td>106</td><td>AY341376.1</td></tr><tr><td>CHIT<sup>1</sup></td><td>Chitin synthase</td><td>Coloration</td><td>CCTTGTGGGATGTCTCCTGAA<br>GCACTAGCTTGCAAAATCGGA</td><td>103</td><td>AB290881.1</td></tr><tr><td>KRMP<sup>1,2</sup></td><td>Lysine (K)-rich mantle protein</td><td>Prismatic formation</td><td>GCCTTCACCACAGAAGGAAG<br>GCCGAATTTCTTCAGACACC</td><td>147</td><td>Ky et al. (2018)</td></tr><tr><td>LINKINE</td><td>Linkine</td><td>Nacre formation</td><td>AGTTGCCAACAGAGTTATACTA<br>TCCACAAAGTTTAATACGAAAA</td><td>104</td><td>EF183520.1</td></tr><tr><td>MRNP34</td><td>Methionine-rich nacre protein 34</td><td>Nacre formation</td><td>GGATACTGTGCTTAGCCGTT<br>TAGACTTTTGTGGCACTGCA</td><td>156</td><td>HQ625028.1</td></tr><tr><td>NACREIN</td><td>Nacrein B4</td><td>Prismatic and Nacre formation</td><td>CAATGAGACCGTCCTTTGGG<br>TTTCTCTGTGTGGGACGTCT</td><td>141</td><td>HQ654772.1</td></tr><tr><td>PEARL<sup>1</sup></td><td>Perline matrix protein</td><td>Nacre formation</td><td>TAACCATGACGTGCACACTT<br>CTATGTCATACGGCAGCCAA</td><td>132</td><td>DQ665305.3</td></tr><tr><td>PEROX<sup>1</sup></td><td>Peroxidas</td><td>Prismatic formation</td><td>GGAAGGAGGGCATTTGTCTT<br>CATCTCTGTCATGCCTCCAG</td><td>135</td><td>HE610394.1</td></tr><tr><td>PIF<sup>1</sup></td><td>Pif177-like protein</td><td>Nacre formation</td><td>AGATTGAGGGCATAGCATGG<br>TGAGGCCGACTTTCTTGGC</td><td>137</td><td>HE610401.1</td></tr><tr><td>PRISM<sup>1</sup></td><td>Prismalin 14 (Prism uncharacterized shell protein)</td><td>Prismatic formation</td><td>AGGATTCTACAGATTTGGCGA<br>TAACAATTGGAGAGGCACGT</td><td>116</td><td>HE610393.1</td></tr><tr><td>SERP<sup>1,2</sup></td><td>Serine protease inhibitor</td><td>Indirect role in biomineralization</td><td>AGGTGTGTACCATTTCTTCTACGG<br>GCAAACATCTCCTCCATCTCC</td><td>1007</td><td>Lemer et al. (2015)</td></tr><tr><td>SHELL<sup>1</sup></td><td>Nacre uncharacterize shell protein 6</td><td>Nacre formation</td><td>TACATGCCCGACAATTGGTT<br>TTCCTTGGTTTCTTCCGCTT</td><td>100</td><td>HE610404.1</td></tr><tr><td>SHEM1B<sup>1</sup></td><td>Shematrin 1</td><td>Prismatic formation</td><td>GTGTGTCCCAGTCTCTTTCC<br>CCACCTCCGTAAATACCACC</td><td>163</td><td>KC505165.1</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 1 Continued</caption><thead><tr><th>Gene name</th><th>Gene description</th><th>Function</th><th>Seq (5'-3')</th><th>Amplicon Length (bp)</th><th>Accession number and citation</th></tr></thead><tbody><tr><td>SHEM5</td><td>Shematrin 5</td><td>Prismatic formation</td><td>TTTGTGTCAGGGTTGTGGAG TGGCTATCATCATCGTTGCA</td><td>156</td><td>HE610376.1</td></tr><tr><td>TYR2A<sup>1,2</sup></td><td>Tyrosinase 2</td><td>Coloration</td><td>GCGGCTCTACTGTCAAATGG<br>CTGGACCTTTCAGGGACTGG</td><td>106</td><td>Lemer et al. (2015)</td></tr><tr><td>18S rRNA<sup>1</sup></td><td>18S ribosomal RNA</td><td>Reference gene</td><td>TTAGTTGGTGGAGCGATTT<br>TAGCGACGGGCGGTGTG</td><td>365</td><td>Larsen et al. (2005)</td></tr><tr><td>EF-1α<sup>1</sup></td><td>Elongation facto-1 alpha</td><td>Reference gene</td><td>CCACGAGTCCTTACCAGAGG<br>TGGATCACTTTTGCTGTCTCC</td><td>115</td><td>Le luyer et al. (2019)</td></tr><tr><td>β-ACT</td><td>Beta actin</td><td>Reference gene</td><td>CCTCACCCCTCAAGTACCCCAT<br>TTGGCCTTTGGGTTGAGTG</td><td>153</td><td>XM-011446513.3</td></tr></tbody></table>

et al., 2019); [2] the nacre layer-related genes including MRNP34, Perline, SHELL, Linkine and Pif-177 (Gervis and Sims, 1992; Samata et al., 1999; Suzuki et al., 2009; Marie et al., 2012; Lemer et al., 2015; Le Luyer et al., 2019); [3] the gene Nacrein involved in the formation of both prismatic and nacre layers (Miyamoto et al., 1996; Yano et al., 2006; Marie et al., 2012); [4] the genes involved in the color of the shell, Tyrosinase2 and Chitin (Lemer et al., 2015) and [5] a gene indirectly involved in biomineralization process, SERP (Ky et al., 2019).

For normalization of the target genes, various housekeeping genes including 18S ribosomal RNA (18S rRNA), Beta actin (β-actin) and Elongation factor 1 alpha (EF-a1) were initially considered as internal reference genes. The 18S gene was shown as the most stable reference gene for qPCR in *P. persica* (Supplementary Figure 1) and was chosen for normalizing the target genes owing to its constitutive expression patterns in mantle tissue. The primers were designed using the Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0; Primer size: 19-23, Tm: 57-62, Product size ranges: 100-170; Table 1). The specificity of the designed primers was checked by primer blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). In addition, the primers for genes CHIT, KRMP and SERP were adopted from previous studies (Table 1). Then all the primer pairs (Table 1) were checked on synthesized cDNA from *P. persica* using normal PCR to ensure their amplification success and consistency.

qRT-PCR amplifications were carried out on a StepOne Real-Time PCR (ThermoFisher Scientific, MA) in final volume of 10 µl included 1 µl cDNA, 5 µl of 2X SYBR green mix (Ampliqon, Denmark) and 0.25 µM of each primer (listed in Table 1). The reaction started with initial denaturation at 94°C for 120 s and then 40 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 20 s followed by gradual thermo scanning (0.3 degree

per step) for melting curve analysis. The RT-qPCR reactions were run in duplicate for each sample. The Ct values were exported after baseline normalization using the Real-Time PCR StepOne Software (ThermoFisher Scientific, MA). PCR efficiencies for each gene were calculated using LinRegPCR (Ramakers et al., 2003). The relative expression of target genes was calculated by $2^{-ΔΔCT}$ method (Livak and Schmittgen, 2001), and then were log2-transformed.

The log2-transformed gene expression data were tested for normality using Shapiro-Wilk test and homogeneity of variance using Leven's test to meet T-test assumptions. T-student test was used to check the source of significant difference in gene expression between the two morphotypes and the comparison of gene expression between black and orange morphotypes in both warm and cool seasons. For those data failed in normality test, the non-parametric analysis, Mann-Whitney test, was used for analysis. Statistical analysis was conducted using SigmaPlot-v11 (version 11).

The principal components analysis (PCA) was carried out in order to transform the variables and indicated the ordination of target genes against independent variables (color and season) using the *fviz_pca* function of the *factoextra* R package (Kassambara and Mundt, 2017). This function provided 95% confidence ellipses for groups within each data set (Houde et al., 2019; Akbarzadeh et al., 2020).

## Results

Two color phenotypes of *P. persica* i.e., orange and black mantle were observed among the specimens sampled in our study in which the orange phenotype was more abundant than the black one. The results of qRT-PCR revealed that a set of genes were differentially expressed between two phenotypes of *P.*

<footnote n="1">¹ These genes are similar to those used in Lemer et al. (2015).</footnote>

<footnote n="2">² These genes are similar to those used in Ky et al. (2019).</footnote>
persica. Among the genes involved in the formation of the prismatic layer, the mRNA expression of ASP, KRMP, SHEM1B and SHEM5 were significantly higher in the orange phenotype compared to those of black morphotype (*P*<0.05). The mRNA expression of PEROX and PRISM showed no significant differences between the two investigated phenotypes (Figure 2A). The results also showed that the gene CALM was

only expressed in the orange phenotype, while 90% of individuals with black morphotype showed no CALM mRNA expression.

All the genes related to nacre layer formation (MRNP34, PEARL, SHELL, LINKINE and PIF), showed significantly higher transcript levels in the orange morphotype compared to the black phenotype (*P*<0.05). The only exception was the PEARL gene in which no transcripts were detected in the individuals

<figure><img src="image_6.png" /><figcaption>FIGURE 2</figcaption></figure>

(A) The box plots of the expression of genes involved in biomineralization process in orange and black phenotypes of the pearl oyster Pinctada persica. (B) The box plots of the expression of genes involved in biomineralization process in black and orange phenotypes of the pearl oyster Pinctada persica, in winter (cool) and summer (warm) seasons. Grey bars represent data for the black phenotype and orange bars represent data for the orange phenotype. Y axis is in the logarithmic scale (gene expression). Error bars indicate standard deviations. The genes CALM, PEARL and CHIT were only expressed in the orange phenotype. Statistical differences between the phenotypes are indicated by asterisks: \*: *P*<0.05, \*: \*: *P*<0.01, \*: \*: \*: *P*<0.001. (C) Canonical plots of the first two principal components of the biomineralization genes for two mantle morphs (black and orange) of the pearl oyster Pinctada persica sampled in winter (cool) and summer (warm) seasons. Principal component analysis (PCA) was performed on the entire dataset. Ellipses represent 95% confidence areas for the groups within treatments. Centroids are represented by the largest point of the same color. Arrows represent loading vectors of the genes.
with black phenotype, while expression of this gene was detected
in 52.3% of individuals with orange mantle type (Figure 2A).

The *NACREIN* gene which is involved in the formation of both prismatic and nacre layers, also showed significantly higher expression in the mantle of orange morphotype compared to the black morphotype (P<0.05; **Figure 2A**). Moreover, among the pigmentation related genes, *TYR2A* showed significantly higher mRNA expression in the orange morphotype compared to the black one (P<0.001; **Figure 2A**). The *CHIT* transcripts were not detected in the black morphotype and only 42.8% individuals of orange phenotype showed *CHIT* transcripts. In addition, no significant differences were observed in the expression of the gene *SERP* between two morphotypes (P>0.05; **Figure 2A**).

The variations of the gene transcripts in both phenotypes
of *P. persica* in each season are shown in Figure 2B. In warm
season, the mRNA expression of *ASP*, *KRMP*, *SHEM5*,
*LINKINE* and *NACREIN* was significantly higher in the
orange phenotype compared to those of black morphotype
(P<0.05; *Figure 2B*), while no significant differences of these
genes were observed between the two phenotypes in winter
(Figure 2B). The *SHEM1B*, *SHELL*, *PIF* and *TYR2A* genes
were significantly higher in the orange phenotype compared
to those of black one in both warm and cool seasons
(P<0.05; *Figure 2B*).

The PCA analysis revealed that the first cleavage factor is
color, with a separation of orange and black groups as shown in
PC1 in Figure 2C. The level of variation range in orange group
was also more important than that of black phenotype. There
was no clear separation between the individuals sampled in two
seasons for either color (Figure 2C).

Discussion

In pearl oysters, the color polymorphism of mantle tissue is
tightly linked to the color and radiance of shell and pearl (Ky
et al., 2019). Therefore, the study of biomineralization and the
genes related to this biological process in mantle tissue of pearl
oysters can be used as a suitable approach to better
understanding the molecular mechanisms that influence shell
and pearl quality and color variations. In this study, we
investigated the transcription of a panel of genes encoding
proteins involved in biomineralization and pigmentation in
black and orange mantle morphotypes of pearl oyster, P.
*persica* in both warm and cool seasons using qRT-PCR. Our
results showed differential expression of genes between two color
morphs of *P. persica*. The major genes chosen for their
involvement in biomineralization of the prismatic and nacre
layer, and pigmentation systematically exhibited a significantly
higher expression in orange compared to the black phenotypes,
suggesting the more active role of these genes in the formation
and the color of shell and probably the pearl in the orange
morphotype of *P. persica*.

Shell coloration has been shown to be related to the original
pigments and the chemical interactions between the pigments
and the shell matrix in mollusks (Williams, 2017). The pigments
may be inserted in the outer or other layers of shells (Budd et al.,
2014; Williams, 2017). The molecular pathway of the melanin
synthesis has an important contribution to the shell and pearl
pigmentation (Palumbo, 2003; Lemer et al., 2015). According to
the results of the present study, the TYR2A gene that is the most
important enzyme in the melanin production (Ding et al., 2021)
was higher expressed in the orange phenotype of P. persica
compared to that of black mantle phenotype. The tyrosinases
play diverse roles in mollusk tissues and shells including shell
matrix formation (Degens et al., 1967; Gordon and Carriker,
1980; Waite, 1983; Feng et al., 2017), pigmentation (Nagai et al.,
2007; Takgi and Miyashita, 2014; Feng et al., 2017), and
producing of the periostracum (Waite and Wilbur, 1976;
Waite, 1983; Feng et al., 2017). It is likely that tyrosinases play
a different role in the formation of the shell (calcitic) layer and
melanin synthesis between two color morphs of P. persica which
may result in different amounts of pigments and pigments
intensity in different phenotypes (Stenger et al., 2021). The
relationship between gene expression and shell pigmentation
was reported in the clam *Meretrix meretrix* with high expression
of the genes in the deeper shell color samples (Yue et al., 2015).
In addition, the relationship between the shell and mantle
pigmentation (Brake et al., 2004) and heritability of shell
pigmentation (Evans et al., 2009) were described in Pacific
oyster, *Crassostrea gigas* (Kang et al., 2013). Opposite to the
findings of Ky et al. (2019) and Lemer et al. (2015), our
results showed significantly differential expression of *TYR2A*
between two morphotypes of *P. persica*. The high expression of
*TYR2A* in orange phenotype may thus be related to the
composition of non-functional melanin protein, a hypothesis
reported for other genes such as Zinc metalloprotease (King and
Summers, 1988; Oetting and King, 1999; Lemer et al., 2015;
Ky et al., 2019).

In addition to tyrosinase, chitin related proteins play
prominent roles in the shell formation of bivalve (Feng et al.,
2017) and melanin biosynthesis (Lemer et al., 2015), however the
expression of CHIT here was only detected in the orange
phenotype, suggesting that an alternative pathway might exist
in the black phenotype of P. persica. Both CHIT and PEARL
genes which are strongly involved in the formation of nacre in
pearl oysters (Samata et al., 1999; Miyashita et al., 2000; Kono
et al., 2000; Suzuki and Nagasawa, 2007; Yan et al., 2007;
Montagnani et al., 2011) and Calmodulin which is prismatic
related gene (Marie et al., 2012) only expressed in orange
phenotype of P. persica. The calcium binding proteins such as
calmodulin, probably play an important role in the regulation of
uptake, transport and secretion of calcium and may be essential
regulators in the extracellular nacrein construction (Miyamoto
et al., 1996; Huang et al., 2007). The MRNP34 may be putatively
involved in protein/protein interactions, by analogy with other
proteins, like calmodulin, which shows two hydrophobic methionine-rich domains that bind to the target protein (Siivari et al., 1995) and involved in nacre formation (Marie et al., 2012). Therefore, the high expression of these genes in orange morph may confirm their interaction that leads to the enhanced biomineralization and the regulation of microstructure of nacreous layer in this phenotype. Taken together, these results show the important roles of these genes in the calcium metabolism during nacre and prismatic layers formation in orange phenotype of *P. persica* compared to the black one, which should lead to different rate of calcification, calcium content and the aragonite and calcite crystals arrangements in the shell and pearl. It is likely that other genes from different pathways or the genes that their function is still unknown play similar roles in the black morphotype of *P. persica*.

The peroxidases are formed of two clusters that could be classified as melanin biosynthesis group and the shell formation group (Feng et al., 2017). In this study, we did not observe different expression of PEROX between the two morphotypes, suggesting this gene may not be a key gene of melanin synthesis in *P. persica*.

Higher expression of *PIF* in orange phenotype of *P. persica* than that of black phenotypes confirmed the distinct functions of this gene between two color morphs. Pif-177 is an important component of the organic matrix for the nacreous layer growth of pearl oysters (Suzuki et al., 2009), consists of aragonite-binding domain, chitin-binding domain, and protein-protein interaction domain (Feng et al., 2017). It is believed that *PIF* is involved in the establishment of color in the shell of *P. margaritifera* (Lemer et al., 2015). Lemer et al. (2015) argued that the lower expression of *PIF* in albino than that of black morphotype of *P. margaritifera*, is involved in their different aragonite crystals arrangement that is associated to lower quality in the colorless nacre layer of albino morphotype (Lemer et al., 2015). Therefore, it can be suggested that the higher expression of *PIF* in orange phenotype of *P. persica* might lead to a higher quality of nacre in this phenotype.

The transcripts of two genes encoding shematin proteins (*SHEM1B* and *SHEM5*) were remarkably higher expressed in orange phenotype of *P. persica* than those of black mantle phenotype. The shematin proteins are the most common proteins synthesized in the mantle edge of mollusks (Yano et al., 2006; Marie et al., 2012). These proteins are secreted into the prismatic layer of the shell, which is thought to provide a template for calcification and are responsible for the hardness of the shell (Yano et al., 2006). These results show the important roles of the shematin family proteins in calcification of different pearl oyster morphotypes that can cause different hardness in shell and pearl.

Aspein shell matrix protein is known as the most acidic protein in mollusk shell matrix protein that controls the contribution of calcite (Tsukamoto et al., 2004). Higher

expression of *ASP* and *KRMP* genes that encode proteins involved in calcite layer formation (Tsukamoto et al., 2004; Zhang et al., 2006) in the orange phenotype of *P. persica* might indicate that the calcite layer framework and arrangement differ in the orange compared to the black phenotypes. In *P. margaritifera*, higher expression of *ASP* and lower expression of *KRMP* was observed in the black compared to albino phenotypes (Lemer et al., 2015), while no differences in the expression of *KRMP* were reported between black and orange mantle of *P. margaritifera* (Ky et al., 2019). Such contrasted expression patterns of genes between closely related species suggest the complex and species specific (and possibly evolutionary convergent) genetic processes are involved in the biomineralization in mantle tissue of pearl oysters. Moreover, both *ASP* and *NACREIN* play important roles in pearl quality and color phenotypes (Blay et al., 2018). It has been reported that the expression of *ASP* is associated with the pearl sac and quality traits (surface defects, lustre and grade) and color in pearl oysters (Blay et al., 2017). Since the high expression of *NACREIN* and *ASP* was observed in orange phenotype of *P. persica* compared to the black one, we expect to observe different quality and color of pearls between two morphotypes.

Matrix proteins regulate the crystal phase, shape, size and nucleation of CaCO3 crystals (Liu et al., 2012). Altogether, differential expression of the nacre and calcite layer-related genes i.e., *ASP*, *KRMP*, *SHEM1B*, *SHEM5*, *LINKINE*, *MRNP34*, *SHELL*, *PIF*, *NACREIN* and coloration (*TYR2A*) between two morphotypes of *P. persica* might indicate the different regulation of these genes and its consequences in the rate of calcification, the design of aragonite and calcite crystals in the shell and pearl, the calcium content, the thickness and weight of the prismatic and nacre layers, intensity and pattern of pigmentation, variability of nacre color of shell and pearl color in oysters with different mantle morphotypes in this species. Some studies investigated the gene expression patterns with different phenotypic traits, which showed differential gene expression between different phenotypic groups (Shi and He, 2014; Lemer et al., 2015; Yue et al., 2015; Blay et al., 2017; Blay et al., 2018; Ky et al., 2018; Ky et al., 2019; Stenger et al., 2021). In contrast to our study, the genes encoding the organic matrix proteins produced by the mantle were found to be highly expressed in the black specimens of *P. margaritifera*. It is stated that the pigmentation-related genes i.e., *SHEM4*, *KRMP*, *MP8*, *CHIT* and *SERP* are involved in the black eumelanin synthesis (Lemer et al., 2015). Moreover, Lemer et al. (2015) reported more expression of genes involved in prismatic layer in the black specimens of *P. margaritifera* and suggested that variation in shell color can primarily occur in the prismatic layer (Lemer et al., 2015). Therefore, the origin of color can result from multiple interactions between both prismatic and nacre layer, and the pigmentation-related genes, which may be conducted by distinct genes in congeneric, even in closely related species.
The beauty and lustre of pearls are related to the surface nacreous layer. The nacre layer is made of CaCO3 aragonite tablets, chitin and matrix proteins (Greigoire, 1957; Watabe, 1965; Wise, 1970). The accurate and orderly assembly of the crystals and organic matrix not only increases lustre by reflecting light uniformly from the layered compartment (Mann, 2001), but also strengthens the nacreous layer to provide fracture resistance (Smith et al., 1999). The highest levels of matrix protein expression is correlated with the ordered deposition of CaCO3 (Liu et al., 2012). According to our result, we might expect a high quality of pearl in orange phenotype of *P. persica* compared to the black one, however experimental approaches are needed to test this hypothesis. Effective selection based on shell and mantle edge color has been already reported in the Pacific oyster (Kang et al., 2013). The selection programs can improve traits such as pearl size, color, darkness, surface defects and grade (Blay et al., 2018).

Comparing two phenotypes of *P. persica* in warm and cool seasons showed that the upregulation of ASP, KRMP, SHEM5, LINKINE and NACREIN genes in orange phenotype was only observed in warm season, but PIF, SHELL, SHEM1B, and TYR2A were significantly upregulated in the orange phenotype in both warm and cool seasons. It is likely that warmer season might modulate higher expression of genes involved in biomineralization process in more dominant phenotype of *P. persica* inhabiting sub-tropical ecosystem of Persian Gulf, and this might affect the quality and color of pearls in orange phenotype of *P. persica*. However, other environmental factors also might affect the expression of biomineralization genes in mantle tissue of pearl oysters. Previous studies demonstrated the responses of biomineralization-related genes to food, ocean acidification and temperature in pearl oysters (Liu et al., 2012; Moya et al., 2012; Joubert et al., 2014; Li et al., 2016). Various studies have showed the effect of temperature on the relative thickness and growth of the shell in oysters (Pouvreau and Prasil, 2001; Kanazawa and Sato, 2008). In the closely related species, *P. margaritifera* inhabiting cooler temperatures, exposing to high temperature caused a decrease in the expression biomineralization genes and negatively impacted shell growth [79]. It has been suggested that the thermal thresholds are probably specific to the ecological limits of the pearl oysters [79]. Further experiments might be needed to explore the possible influence of inhabiting warmer temperatures on the nature of nacre and the possible evolution of dominant phenotypes, and possibly the potential for high quality pearl production in natural populations of *P. persica*.

## Conclusion

The goal of present study was to compare the expression of genes involved in biomineralization and pigmentation in the

orange and black phenotypes of *P. persica*. Our results showed different expression profiles of genes between these two color morphs. Major genes involved in biomineralization and pigmentation (except for PEROX and PRISM) were significantly higher expressed in the orange phenotype of *P. persica* that is more dominant morphotype in Persian Gulf, which is contrary to the results obtained in the closely related species, *P. margaritifera* with higher expression of biomineralization and pigmentation genes in the black dominant morphotype (Lemer et al., 2015; Ky et al., 2019).

## Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

## Ethics statement

This study was approved by Qeshm Department of Environment of QFAO.

## Author contributions

MR supervised the study and conducted the sampling. MR and AF designed the experiments. FP, AF and MR carried out the molecular genetic studies and experiments in the laboratory and wrote the manuscript. AA, MR, FP and SA-H analyzed the data and contributed to reviewing the manuscript. All authors contributed to the article and approved the submitted version.

## Funding

This study was financially supported by Persian Gulf Biotechnology Park (PGBP), Qeshm Island, Iran grant no. 431.98.

## Acknowledgments

Especial thanks go to Kaveh Khosraviani (CellKGP Company, Genetic and Biotechnology laboratory, Karaj, Iran) for laboratory and technical support. We are also grateful to Persian Gulf Biotechnology Park (PGBP) for their generous support to develop this study. The authors also would like to acknowledge the support provided by Eghbal Zobeiri for the map preparation. All individual rights, animal rights and scientific ethics have been observed in this study.
## Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated

organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

## Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2022.1038692/full#supplementary-material

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