﻿Intersexuality in a natural population of the terrestrial isopod Porcellioscaber

﻿Abstract Intersex phenotypes are rarely observed in natural isopod populations and their expression is typically associated with infection of Wolbachia, a reproductive parasite that manipulates arthropod reproduction. During an intensive sampling effort of a natural population of the isopod Porcellioscaber, an adult individual was isolated that expressed both male and female traits. The intersex individual exhibited clearly developed external male genitalia and carried multiple eggs in its brood pouch. No Wolbachia infection could be identified in this individual, a result that needs to be approached with caution due to suboptimal DNA preservation for diagnostic PCR assays. Wolbachia were, however, detected in two adult females of the same population, and appear closely related to isolates that infect other terrestrial isopod species. This is the first demonstration that intersex phenotypes can arise under natural conditions in P.scaber.


Introduction
Sex-determination mechanisms regulate the sexual differentiation of organisms and are highly diverse across the animal kingdom. Sex-specific differentiation can rely on external environmental cues but can also be solely regulated by the segregation of genetic factors (Bull 1985;Zarkower 2001). In a wide range of arthropod species, sexdetermination is controlled by multiple genetic factors (Cordaux et al. 2011;Moore and Roberts 2013). For instance, populations of the house fly Musca domestica carry several male-determining chromosomes and an additional female sex-determination locus (Dübendorfer et al. 2002;Hediger et al. 2010). Intersex individuals that express both male and female traits spontaneously arise at low frequencies in natural populations of certain arthropod species (Narita et al. 2010). In isopods, the appearance of intersex individuals is often linked to infection of Wolbachia, probably one of the most widespread invertebrate-associated bacteria (Bouchon et al. 1998(Bouchon et al. , 2009Engelstädter and Hurst 2009;Cordaux et al. 2011Cordaux et al. , 2012. Wolbachia are maternally transmitted endosymbiotic bacteria that infect the reproductive tissues of arthropods and nematodes (Werren 1997). Wolbachia spread within host populations by manipulating host reproduction in multiple ways (Engelstädter and Hurst 2009). In isopods, Wolbachia can feminize genetic males into functional phenotypic females (Rigaud et al. 1991;Bouchon et al. 1998;Cordaux et al. 2004). However, under certain conditions, Wolbachia-mediated feminization can be incomplete and intersex individuals can arise (O'Neill et al. 1997;Herran et al. 2020).
To date, Wolbachia-infected individuals have been described in at least 39 isopod species, with all Wolbachia isolates belonging to the Wolbachia B-or F-supergroup (Bouchon et al. 1998;Cordaux et al. 2001;Zimmermann et al. 2021). Wolbachiamediated feminization of the common pill woodlouse Armadillidium vulgare is well understood and has become a model system to study the evolution of sex-determination pathways (Rigaud et al. 1997;Herran et al. 2020). Here, the androgenic hormone synthesized by the androgenic gland is responsible for the formation of male gonads. It has been experimentally shown that the implantation of an androgenic gland into females can induce functional sex reversal depending on the timing of implantation (Suzuki and Yamasaki 1997). These findings indicate that all A. vulgare individuals possess the necessary genetic basis that is required for male and female sexual differentiation (Rigaud et al. 1997).
In genetic males, Wolbachia likely inhibit the development of the androgenic gland by either targeting the androgenic hormone promotor or the androgenic hormone receptor hereby feminizing the individual (Rigaud et al. 1997). Incomplete feminization has been associated with low densities of Wolbachia during embryonal development (O'Neill et al. 1997;Bouchon et al. 2009;Cordaux et al. 2011;Herran et al. 2020). Low Wolbachia densities can be the result of increased temperature levels as has been shown for A. vulgare (Herran et al. 2020). Despite the widespread occurrence of Wolbachia in natural isopod populations (Bouchon et al. 1998(Bouchon et al. , 2009), intersex individuals have been observed in only a limited number of isopod species, including A. vulgare, Armadillidium album, Porcellio laevis, Sphaeroma rugicauda and Ligia oceanica Bouchon et al. 1998;Ben Nasr et al. 2010).
Here, we present the first record of an intersex Porcellio scaber collected from a natural population in Snellegem (Belgium) in August 2020. The individual carried a large number of eggs in its brood pouch, and can thus be considered as a functional female. However, the individual also possessed clearly developed external male genitalia.

Isopod collection
We used cuboid pitfalls (25 cm × 7.5 cm × 8 cm) containing an approximately 1 cm bottom layer of plaster to collect isopods in Brugge, Snellegem, and Vleteren (Parmentier et al. 2021). The plaster was moisturized to prevent the desiccation of all trapped isopods, so that we could collect the isopods alive; all isopods were collected one week after installation of the pitfalls and preserved on 70% ethanol. Individuals were preserved on 70% ethanol for three months before phenotyping.

Diagnostic PCR detection of Wolbachia infection
In addition to the intersex individual, we also isolated four adult females that exhibited normal sexual differentiation from the Snellegem population. Sterility was maintained by working in a biological safety cabinet. After washing the specimens twice in sterile water for 1 min, DNA was extracted from whole bodies using the Quick-DNA Universal kit (BaseClear, the Netherlands). DNA integrity was tested by amplifying a fragment of cytochrome c oxidase subunit I (COI) using the LCO1490 and HC02198 primers (Folmer et al. 1994). A standardized PCR approach was performed to test Wolbachia infection in the five P. scaber samples (one intersex individual and four normal females) using DreamTaq DNA Polymerase (Life Technologies Europe BV). The standard primers of the multilocus sequence typing system for Wolbachia were used to amplify fragments of the wsp, ftsZ, hcpA, coxA, and gatB genes, standard molecular markers to detect Wolbachia infection (Baldo et al. 2006). Wolbachia-infected Myrmica scabrinodis workers were used as positive controls. The hcpA gene fragment was Sanger sequenced for one of the Wolbachia-infected P. scaber females that exhibited normal sexual differentiation (MACROGEN Europe B.V.).

Results
From a collection of 7,814 individuals, we found a P. scaber individual in the Snellegem population that carried eggs and, although egg viability was not ascertained, could be considered as a functional female (Fig. 1). However, in contrast to female isopods ( Fig. 2A), this individual possessed an elongated and well-developed endopodite of the first pleopod, a canonical male sexual characteristic in isopods (Fig. 2C). In addition, the exopodites and endopodites of the first and second pleopods were also shaped differently than females, forming a male pleon (Fig. 2B, C). In the marsupium of the individual multiple eggs were found, comparable in number to normal females. We did not manage to amplify a fragment of the COI gene for the intersex individual, while PCR amplification was successful for four normal Snellegem females that were collected at the same time. This apparent discrepancy in DNA integrity might be due to a longer exposure to air for the intersex individual during photography. Using  diagnostic PCR assays, we were unable to identify a Wolbachia infection of the intersex individual. Wolbachia infection was confirmed in two of the four normal females. Sanger sequencing of the hcpA gene fragment revealed a 100% identity to a Wolbachia isolate that was previously retrieved from the isopod Helleria brevicornis (Sicard et al. 2014). The partial hcpA gene sequence was deposited in the GenBank database under the accession number OM459769.

Discussion
Intersex individuals are rarely observed in natural populations of arthropods (Narita et al. 2010). The intersex phenotype of the current study appears to be present at an extremely low frequency in natural populations of P. scaber. However, additional intersex individuals were possibly overlooked, because they were not carrying visible eggs at the time of collection. One study previously described a similar, but not identical, intersex phenotype in P. scaber where three specimens displayed greatly reduced male genitalia and were expected to be functional females according to the authors (Sassaman and Garthwaite 1984). In contrast to our individual, these individuals came from laboratory stocks and not from a wild natural population. In addition, our individual did not show reduced genitalia.
It is tempting to speculate that incomplete Wolbachia-mediated feminization caused the intersex phenotype in this individual. Wolbachia are widespread in P. scaber, infecting populations across Europe (Bouchon et al. 1998). We uncovered that the natural Snellegem population was also infected. Previous work has shown that both males and females of P. scaber carry Wolbachia (Bouchon et al. 1998). Moreover, interspecific transfer of feminizing Wolbachia into P. scaber revealed that its sex-determination mechanisms can be manipulated by the reproductive parasite and can result in intersex individuals under controlled laboratory conditions (Bouchon et al. 1998). However, it remains uncertain whether the Wolbachia variants that naturally infect P. scaber are able to feminize genetic males. Unfortunately, we could not bring more clarity to this outstanding question due to our inability to confirm Wolbachia infection in our intersex individual. Due to the suboptimal preservation of the individual (Marquina et al. 2021), DNA degradation was likely too severe and interfered with our diagnostic PCR assays, a hypothesis that is supported by our inability to amplify a COI gene fragment. Currently, we cannot exclude that other mechanisms, such as pollutionor virus-induced developmental abnormalities, caused the formation of a functional female P. scaber with male genitalia Ford 2012).