Bacteria in spiders? Presence of Wolbachia (Rickettsiales, Ehrlichiaceae) in Synspermiata spiders, including the first record for the family Sicariidae (Araneae)

¿Bacterias en arañas? Presencia de Wolbachia (Rickettsiales, Ehrlichiaceae) en arañas Synspermiata, incluyendo el primer registro para la familia Sicariidae (Araneae)

Claudia Isabel Navarro-Rodríguez

Universidad Autónoma de Tlaxcala, Mexico

Alejandro Valdez-Mondragón lat_mactans@yahoo.com.mx

Centro de Investigaciones Biológicas del Noroeste, Mexico

Alma Rosa Juárez Sánchez

Autor Independiente, Mexico

Samuel Nolasco

Autor Independiente, Mexico

WolbachiaHertig & Wolbach, 1924, is a common genus of intracellular endosymbiotic bacteria belonging to the order Rickettsiales (family Ehrlichiaceae) that is known to infect a wide variety of animal groups including arthropods, such as insects, crustaceans, and arachnids (Hertig, 1936; Werren, 1997). The presence of Wolbachia has been reported in approximately 40% of terrestrial arthropods, mainly insects, likely making it the most widespread endosymbiont found on earth (Fenn et al., 2006; Zug et al., 2012; Yang et al., 2021). As was mentioned by Yang et al. (2021) within arthropods, Wolbachia has previously been found in isopods (Zimmermann et al., 2015), mites (Chen, 2016), scorpions (Baldo et al., 2007), spiders (Dunaj et al., 2020; Goodacre et al., 2006), and insects (Hilgenboecker et al., 2008; Yang et al., 2013, 2021). It is estimated that around 66% of insect species are infected with this bacterium (Hilgenboecker et al., 2008), making it one of the most reported reproductive parasites in spiders (Goodacre, 2011).

Within arthropods, this intracellular bacteria can manipulate host reproduction in multiple ways in order to favor its own reproduction, such as: 1) inducing parthenogenesis, 2) increasing the proportion of females (killing male offspring), 3) feminization and cytoplasmic incompatibility, thus promoting its survival, and 4) spread (Durkin et al., 2021; Goodacre, 2011; Vanthournout et al., 2011; Yang et al., 2021). The main transmission strategy usually occurs through vertical transfer from the mother to the offspring through the cytoplasm of the ovule (Werren, 1997), the wide distribution of this bacterium is difficult to explain solely by vertical transmission, there is also evidence of horizontal transfer through the food chain by sharing common food sources, as well as by parasites such as infected mites or wasps (Ahmed et al., 2016; Le Clec'h et al., 2013; Su et al., 2019).

Wolbachia infections in spiders have been found in 99 species across 62 genera and 17 families (Yang et al., 2021). This infection has been reported in both entelegynes spiders and Synspermiata spiders (= Haplogynae) (Goodacre, 2011). The most complete study of Wolbachia in spiders was carried out by Yang et al. (2021), who collected 1153 spiders from China and screened for Wolbachia in 975 individuals. However, to date there have been no records of Wolbachia in the spider family Sicariidae, including the genus Loxosceles that is medically important for humans due to their venomous bites (Vetter, 2015).

Although the bacteria Wolbachia has been recorded in wide spectrum of arthropods including arachnids worldwide, it has never been recorded previously for spiders from Mexico, which represent an opportunity to know its endosymbiotic interaction with spiders from Nearctic and Neotropical regions of the country.

In this study, we documented for the first time the presence of Wolbachia in the spider genus Loxosceles (Sicariidae) as well as in the genus Physocyclus (Pholcidae) based on molecular evidence from the mitochondrial Cytochrome c oxidase 1 (CO1) locus. These findings represent the first records of the bacteria Wolbachia in spiders from Mexico.

Spider collection and DNA extraction. This study was performed in parallel with a project on species delimitation in Physocyclus and Loxosceles spiders from Mexico using morphological and molecular data (Nolasco & Valdez-Mondragón, 2022; Valdez-Mondragón et al., 2019; Navarro-Rodríguez & Valdez-Mondragón, 2024). Spiders were collected by hand between 2017 and 2019 from different localities and states across Mexico. Specimens were preserved in 80% ethanol for species identification and 96% ethanol for molecular studies, along with their collection data and identification to species level. Tissue selection for molecular analyses depended on the available material and the size of the specimens but was mainly isolated from whole legs (juveniles and adults), while the prosoma and opisthosoma were occasionally used in juveniles, following the protocols by Navarro-Rodríguez and Valdez-Mondragón (2020, 2024). Once the tissue was separated, it was stored in 96% ethanol under refrigeration at -20°C for subsequent extraction using a Qiagen DNeasy® extraction kit following the extraction protocols and modifications for pholcids spiders (Physocyclus) by Nolasco and Valdez-Mondragón (2022) and for violin spiders (Loxosceles) by Navarro-Rodríguez and Valdez-Mondragón (2020, 2024).

Amplification and sequencing. The mitochondrial marker Cytochrome c oxidase subunit I (CO1) was amplified using the following primer sets: LCO1490/HCO2198 and LCO1490-JJ/HCO2198-JJ (Folmer et al., 1994; Astrin & Stüben, 2008) (Appendix 1). The PCR (Polymerase Chain Reaction) parameters for CO1 were performed following the protocols by Navarro-Rodríguez and Valdez-Mondragón (2024). The sequencing of both forward and reverse chains (5′-3′ and 3′-5′) of the PCR products was performed in a Sequencer Genetic Analyzer RUO Applied Biosystems Hitachi model 3750xL at the Laboratory of Molecular Biology and Health, IB-UNAM, Mexico City.

DNA sequence editing and BLAST. BioEdit v. 7.0.9.0 (Hall 1999) and Geneious v. 8.1.9 (Rozen & Skaletsky, 2000) were used for molecular sequence editing. Once the sequences were edited, they were verified using the BLAST tools (www.ncbi.nih.gov/Blast.cgi) to find regions of similarity between the newly obtained sequences and those deposited in the GenBank database (www.ncbi.nih.gov), as well as to identify possible contamination. The parameters used under BLAST were as follows: Choose Search Set: Database (Standard databases (nr etc.), nucleotide collection (nr/nt); Program selection: Optimize for Highly similar sequences (megablast); Algorithm parameters (General parameters, Scoring Parameters, Filters and Masking): standard.

According to the best matches from the BLAST nucleotide search, Wolbachia bacteria was identified in a total of 29 specimens belonging to eight species of spiders within the families Pholcidae and Sicariidae. Specifically, Wolbachia was found in four sequences from two individuals of Physocyclus hoosgtrali Gertsch & Davis, 1942 (Pholcidae) collected from two different populations in Nuevo León in northeast Mexico; as well as in 25 sequences from 17 individuals of seven different Loxosceles species from different localities and states across Mexico (Table 1). Of the samples sequenced, seven corresponded to adult male spiders, five to adult females, and seven to juvenile specimens. Wolbachia was mainly found in tissues from males, females, and juveniles, but also from complete juvenile specimens.

Table 1
Spider species of Physocyclus (Pholcidae) and Loxosceles (Sicariidae), sequence vouchers, and localities in Mexico used for DNA extraction and PCR of the molecular marker CO1, indicating the tissue used for molecular studies. The samples include only those recorded with the bacteria Wolbachia. HCO= reverse, LCO= forward.

BLAST searches of CO1 and CO1-JJ found high Query Coverage matches with Wolbachia bacteria (>90%) for 19 sequences, and low Query Coverage matches (<90%) in 9 sequences (Table 2). Regarding the Highest percent identity, 15 sequences had >90% identity, whereas 15 sequences showed <90% identity (Table 2). Fourteen sequences corresponded to Wolbachia pipientisHertig, 1936, including all sequences of P. hoogstraali, whereas 15 sequences from Loxosceles corresponded to Wolbachia spp.

Table 2
Summary of the sequences from Physocyclus (Pholcidae) and Loxosceles (Sicariidae) found with the highest significant alignment identities and similarities with the bacteria Wolbachia using the BLAST search tools. HCO= reverse, LCO= forward.

Considering the reports of spiders infected by Wolbachia presented in Yang et al. (2021: supplementary information), the results presented herein increase this number to 19 families, 70 genera, and 122 species (Fig. 1). Linyphiidae contains the highest number of species reported to be infected by Wolbachia to date, with 27 genera and 43 species (Fig. 1).

Figure 1
Total number of families, genera, and species of spiders with reports of Wolbachia infections. Updated from Yang et al. (2021: supplementary information) and based on the present work.

Knowledge of the presence of Wolbachia in arthropods has increased in recent decades, as Wolbachia infection has been recorded in 66% of insect species (Hilgenboecker et al., 2008; Zug et al., 2012), 45% of mite species (Breeuwer & Jacobs, 1996; Chen, 2016), and 35% of terrestrial isopod species (Bouchon et al., 1998; Cordaux et al., 2001; Zimmermann et al., 2015). In the case of spiders, the work by Yang et al. (2021) from Mangshan, China, found Wolbachia in the 84.6% of 1153 individuals which represents 68 spider species in 45 genera and 16 families in this study.

According to Yang et al. (2021), exists a phylogenetic incongruence between the phylogenies of the host spiders and their corresponding Wolbachia species, indicating that horizontal transmission may occur between spider species. The four known mechanisms for horizontal transmission are blood contact (Rigaud & Juchault, 1995), host-parasitoid association (Ahmed et al., 2015; Batista et al., 2010), feeding relationship (Kittayapong et al., 2003), and common usage of the same plant tissues (Li et al., 2017; Yang et al., 2013).

Based on our results from studying Synspermiata species, the presence of Wolbachia in P. hoogstraali and seven species of Loxosceles from Mexico is likely due to horizontal transfer and no differences were found between sexes or populations in the studied species, as Wolbachia was

recording in adult males and females, as well as juvenile specimens. Although the exact mechanism of horizontal transmission between spiders remains unknown and requires further research, the blood contact, host-parasitoid association, and feeding relationship are plausible pathways that would allow for horizontal transfer of Wolbachia between spiders (Yang et al., 2021).

According to the results of the BLAST search performed in this study, 14 sequences of CO1 matched with W. pipientis, the type species of the Wolbachia genus that was originally discovered within the ovaries of the mosquito Culex pipiens (Hertig & Wolbach, 1924). The other 15 sequences from Loxosceles corresponded to Wolbachia sp., an endosymbiotic bacterium of Polyergus mexicanus Forel, 1899, a species of slave-making ant in the subfamily Formicinae.

A wide range of bacterial species have been identified to infect different species of spiders (Goodacre, 2011), even within the Synspermiata spiders (Cordaux et al., 2001; Rowley et al., (2004); despite this, this study represents the first record of Wolbachia infection in the genus Loxosceles and more broadly in the family Sicariidae. While most studies exhibit a predilection for recognizing the effects of this bacterium in females (Vanthournout et al., 2011; Vanthournout & Hendrickx, 2015), we found Wolbachia to be present mostly in male tissues. The implications that this infection carries in both males and females remains to be fully understood, and the carriers of this bacterium may convey certain advantages, as mentioned in previous works (Correa & Ballard, 2016). Similarly, an in-depth study on the types of bacteria that could infect Physocyclus and Loxosceles species is needed, since at least four other endosymbionts have been found alongside Wolbachia in other spider species (Vanthournout et al., 2011).

Finally, combining our results with the list presented in Yang et al. (2021: supplementary information) increases the number of spider families within the Synspermiata spiders for which Wolbachia infections are known to four families: Dysderidae, Pholcidae, Telemidae, and Sicariidae, now with Pholcidae. The last having the largest number of known infected species in this group, found in four species of the genus Pholcus C. L. Koch, 1850, as well as Physocyclus hoogstraali (Appendix 2). Although Yang et al. (2021) included Atypus suiningensis Zhang, 1985 (Atypidae) in their study on the presence of Wolbachia in different spiders from Mangshan, China, only araneomorphs (Araneomorphae) spiders species have been reported with Wolbachia infections so far.

In conclusion, although Wolbachia was found in 29 specimens belonging to eight spider species of Synspermiata, more additional samples, and even reproductive, ecological and molecular studies are needed to establish whether of such as populations the infections by horizontal transfer is due to feeding relationships or host-parasitoid associations, this last commonly found in spiders (Foelix, 2011; Takasuka & Broad, 2024), without discard the vertical transfer in spiders.

The first author thanks the Posgrado en Ciencias Biológicas, Universidad Autónoma de Tlaxcala (UATx) for educational support, and CONACyT for scholarship support during their PhD in Biological Sciences. The second (AVM) author thanks the program Cátedras CONACyT, Mexico, for scientific support of the project No. 59: “Laboratorio Regional de Biodiversidad y Cultivo de Tejidos Vegetales (LBCTV) del Instituto de Biología, Universidad Nacional Autónoma de México (IBUNAM), sede Tlaxcala”. AVM also thanks SEP-CONACyT for financial support of the project Basic Science (Ciencia Básica) 2016, No. 282834. We also thank the students of the Laboratory of Arachnology (LATLAX), IBUNAM, Tlaxcala, for their help in the field and processing material in the laboratory. We thank Brett O. Butler for the English language review of the manuscript, and the reviewers for their comments and suggestions that improved the quality of the work. All specimens were collected under Scientific Collector Permit FAUT-0309 from the Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT) issued to AVM.