Mitochondrial dna variability and wolbachia infection in two sibling woodlice species

ABSTRACT Several morphological races and subspecies have been described and later included within the terrestrial isopod species _Porcellionides pruinosus_. During our study of this species,

we have worked on specimens from France, Greece, Tunisia and Réunion island. Laboratory crosses have revealed two separate groups of populations: French populations (four localities) in one

group, and those from Tunisia, Réunion island and Greece in the other. French individuals were reproductively isolated from those of the other populations. We have undertaken a survey of

mitochondrial DNA (mtDNA) polymorphism in these seven populations. We observed two groups of mitotypes corresponding to the two groups of populations. Interfertility experiments between

populations and the mitochondrial genetic distances between mitotypes both suggest the presence of two different species, one in France and one in Greece, Tunisia and Réunion island. The two

species harbour, respectively, two different _Wolbachia_ lines. Another feature of the molecular genetic analysis was the apparent mitochondrial monomorphism in the French populations and

the low variability in the other three populations. The result can be related to the possibility of _Wolbachia_-induced genetic hitchhiking in these populations. SIMILAR CONTENT BEING VIEWED

BY OTHERS PHYLOGEOGRAPHIC ANALYSES REVEAL RECENT DISPERSAL AND MULTIPLE _WOLBACHIA_ INFECTIONS OF THE BRIGHT-EYED RINGLET _EREBIA OEME_ WITHIN THE EUROPEAN MOUNTAIN SYSTEMS Article Open

access 14 January 2025 PRONOUNCED MITO-NUCLEAR DISCORDANCE AND VARIOUS _WOLBACHIA_ INFECTIONS IN THE WATER RINGLET _EREBIA PRONOE_ HAVE RESULTED IN A COMPLEX PHYLOGEOGRAPHIC STRUCTURE

Article Open access 25 March 2022 _WOLBACHIA_ AFFECTS MITOCHONDRIAL POPULATION STRUCTURE IN TWO SYSTEMS OF CLOSELY RELATED PALAEARCTIC BLUE BUTTERFLIES Article Open access 04 February 2021

INTRODUCTION _Porcellionides pruinosus_ is assumed to be the most widely distributed terrestrial isopod across the world (Vandel, 1960). However, this species has had a complex and confused

taxonomic history. Because of their morphological variation, specimens of _P. pruinosus_ from Europe and from Africa have been given subspecific rank (Verhoeff, 1918; Strouhal, 1938).

Additionally, Garthwaite & Sassaman (1985) in their studies of North American terrestrial isopods described a new species, _Porcellionides floria_, for specimens previously included

within _P. pruinosus_. They suggest that _P. pruinosus_ (as formerly defined) may consist of a number of distinct and localized species rather than one unique cosmopolitan species. We have

undertaken a survey of _P. pruinosus_ individuals from seven different localities. Different approaches give congruent results, showing the existence of two sibling species. In parallel with

the survey of mitochondrial polymorphism, we have investigated the intensity of _Wolbachia_ infection in _P. pruinosus_. The intracytoplasmic _Wolbachia_ bacterium has been found in several

species of insects, in crustacean isopods, in mites and in nematodes (Rousset et al., 1992; Bandi et al., 1998). These maternally inherited microorganisms cause cytoplasmic incompatibility

in many insects, parthenogenesis in _Trichogramma_ wasps and feminization of genetic males in terrestrial isopods (review in Werren & O’Neill, 1997). The isopod suborder Oniscidea

(woodlice) is strongly affected by the presence of these endocytoplasmic bacteria (Bouchon et al., 1998). Their effect on the control of sex determination has been well studied in

_Armadillidium vulgare_, where _Wolbachia_ is believed to inhibit the activity of ‘male genes’ by suppressing androgenic gland development (Martin et al., 1973; Juchault et al., 1993).

Infected eggs develop into functional females, that can transmit bacteria, regardless of their sex chromosome composition (review in Rigaud, 1997). The main consequence of this cytoplasmic

sex determination is distortion of the sex ratio, with infected females producing highly female-biased broods. The same phenomenon also occurs in the woodlouse _P. pruinosus_ (Juchault et

al., 1994; Rigaud et al., 1997). In the present study, we found no mtDNA polymorphism in the populations entirely infected by the feminizing _Wolbachia_, but a small amount of variation in

mtDNA in populations where _Wolbachia_ infection was incomplete. This is consistent with a reduction in mitochondrial diversity resulting from hitchhiking following the spread of the

intracytoplasmic symbiont. MATERIALS AND METHODS SAMPLING The four French populations investigated were from the following locations: Mignaloux (Vienne), Nevers (Nièvre), Poitiers (Vienne)

and St Martin-du-Fouilloux (Deux-Sèvres). All gravid females collected from the wild were reared in the laboratory (20°C; LD: 18:6) and sex-ratios of their progenies noted. Specimens from

Réunion island, Indian ocean (at St Paul), Greece (Athens) and Tunisia (Tunis) were reared in the same conditions. A scanning microscope survey of cuticular ornaments revealed that

individuals from these populations conformed morphologically to the description of the species _P. pruinosus_ (Vandel, 1960; Hadley & Hendricks, 1985) (results not shown). INTERFERTILITY

EXPERIMENTS Crosses between the different French locations were fully interfertile (data not shown). To test interfertility between individuals from distant locations, males and females

from France (St Martin-du-Fouilloux), Greece, Tunisia and Réunion island were combined to obtained 12 interpopulation crosses and four intrapopulation crosses (Table 1). In each crossing

type, eight couples of one male and one virgin female were mated under laboratory conditions (20°C, LD 18 : 6). MITOCHONDRIAL DNA ANALYSIS The total mitochondrial DNA polymorphism was

analysed by restriction fragment length polymorphism. As mitochondrial DNA is maternally inherited, offspring from all broods produced by each mother were pooled in isofemale lines. Total

mitochondrial DNA was extracted from gonads, nervous tissue and fat body, according to Souty-Grosset et al. (1992). The mtDNA was then digested with the following restriction enzymes:

_Kpn_I, _Acc_II, _Cla_I, _Bgl_II, _Pvu_II, _Hin_cII, _Sca_I, _Eco_RI, _Bst_EII, _Sst_I. Most of them recognize six-base sequences, except _Acc_II, which recognizes a four-base sequence. The

digested DNA patterns were run on agarose gels under the conditions described in Grandjean et al. (1993) and fragment migration patterns were visualized by staining with SYBR Green I FMC

(BioProducts). This sensitive fluorescent stain detected as little as 60 pg nucleic acid per band (using 300 nm transillumination). The number of nucleotide substitutions per site, _d_, was

estimated by the method of Nei & Tajima (1983). In addition, a PCR-RFLP survey was made on the mitochondrial 16S rDNA (mt 16S rDNA). The total DNA was extracted from each individual’s

gonads, fat tissue and nervous system, according to Wilson et al. (1985). Amplification of the mt 16S rDNA was carried out according to Kocher et al. (1989) on a thermocycler Trio

thermoblock (Biometra). The mixture contained the two primers, 16 SAR and 16 SBR, that targeted the mitochondrial 16S ribosomal RNA genes (Simon et al., 1991), and _Taq_ polymerase (Promega

Biotech). PCR conditions were described in Bouchon et al. (1994). Amplified portions of mt 16S rDNA were purified by the chloroform method and digested with the following 11 restriction

endonucleases: A_cc_II, _Hha_I, _Msp_I, _Rsa_I, _Hae_III, _Dra_II, _Ssp_I, _Nde_II, _Taq_I, _Hin_fI and _Alu_I. Digested amplification products were run on 1.5% agarose gel in TBE buffer for

45 min at 100 V. After staining with ethidium bromide, fragments were visualized with UV light. DETECTION OF _WOLBACHIA_ MICROORGANISMS The prevalence of _Wolbachia_ has previously been

investigated in the Mignaloux and Nevers populations (Rigaud et al., 1997). The prevalence of _Wolbachia_ in the populations from Poitiers, St-Martin-du-Fouilloux, Réunion island, Greece and

Tunisia, was determined in wild-caught mothers, when possible, or in two offspring. The bacteria were detected by PCR amplification of part of the bacterial 16S ribosomal DNA gene, using

specific primers (99f and 994r) amplifying a 900-bp fragment (O’Neill et al., 1992). The total DNA extraction from the gonads and the PCR conditions were as described in Bouchon et al.

(1998). Amplification products were run on a 1.5% agarose gel in TBE, stained with ethidium bromide and visualized under UV light. From two _Wolbachia_ strains (Réunion island and Poitiers),

we amplified a fragment of 16S rDNA gene homologous to positions 8–973 of the _E. coli_ sequence, by using 27f and 973r primers (Rousset et al., 1992). From amplification products, direct

double strand sequencing was performed as described by Bouchon et al. (1998). The primers used for sequencing were 27f, 685r, 530f and 973r (Rousset et al., 1992). Using these primers, 812

nucleotide sequences of the rRNA gene were determined on the both strands. The GenBank, EMBL and DDBJ accession numbers for the nucleotide sequences of the two isolate of _Wolbachia_ are

AJ133196 (Réunion island) and AJ223242 (Poitiers). RESULTS INTERFERTILITY ANALYSIS As shown in Table 1, there was no interbreeding between French specimens and the others. In these series of

crossing no mating behaviour was observed. The reproductive isolation was therefore prezygotic, which was confirmed by the fact that no sperm was found in the females’ genital tracts after

the experiment. In comparison, individuals from Réunion island, Greece and Tunisia were totally interfertile. Throughout, no distinguishable morphological characters were detected,

especially on the male copulatory appendix. MITOCHONDRIAL DNA ANALYSIS The _P. pruinosus_ mtDNA was atypical in size, around 42 kb. The size, and the restriction patterns, were similar to

those of the mtDNA from another terrestrial isopod _A. vulgare_ (Raimond et al., 1999). The mtDNA seems to be composed of three identical 14 kb monomers. One of these is linear, whereas the

other two form a circle, with one monomer reversed with respect to the other. A single restriction site per monomer generates a four-band pattern in a restriction profile and additional

restriction sites generate three identical fragments leading to a single band (Raimond et al., 1999). These fragments have to be counted triply to obtain a correct estimation of the size of

the mtDNA (Table 2). The RFLP analysis revealed three mitotypes: M1, M2 and M3. There was no mtDNA polymorphism in the four French populations, all the isofemale lines (20 for St Martin,

nine for Poitiers, 10 for Nevers and 11 for Mignaloux) having the same mitotype, M1. Mitotype M1 was strictly confined to the French populations and was not found in the Réunion island,

Greek and Tunisian populations, whereas M2 and M3 were shared by these last three populations. All of the enzymes exhibited a distinct profile between the mitotype M1 and the two mitotypes

M2 and M3. The M2 and M3 mitotypes were closely related (_d_=0.0092). M1 was much more distantly related to each of them (_d_=0.4448 between M1 and M2; and _d_=0.4490 between M1 and M3). The

PCR-RFLP analysis of the mitochondrial ribosomal gene revealed two distinct mitotypes, one for the four French populations and one for the three others. As shown in Table 3, six restriction

enzymes out of the 11 distinguish the French mitotypes from the others: _Alu_I, _Msp_I, _Hae_III, _Nde_II, _Hin_fI and _Dra_II. We obtained identical restriction patterns for Tunisian,

Greek and Réunion island individuals. The nucleotide distance estimated according to Nei & Tajima (1983) was 0.133 between the mitotype harboured by the French population and the

mitotype harboured by the Réunion island, Tunisian and Greek populations. SEX RATIOS, _WOLBACHIA_ INFECTION AND RELATIONSHIP WITH MTDNA VARIABILITY Analysis of progeny sex ratios showed that

females infected with _Wolbachia_ produced female-biased offspring, whereas those that were not infected produced a more balanced sex ratio (Table 4). The differences in the sex ratios of

offspring from infected and uninfected females were highly significant in Tunisia (χ24=26.22; _P_ < 0.01) and Réunion (χ27=60.41; _P_ < 0.01), as had been seen in Nevers (Rigaud et

al., 1997). _Wolbachia_ studies in French populations (Rigaud et al., 1997) showed that almost all females in the wild harboured _Wolbachia_, with one population, Mignaloux, being 100%

infected. Only one uninfected female was found in Nevers (Table 5). Surveys of the Poitiers and St-Martin-du-Fouilloux populations showed that all the females tested were infected with

_Wolbachia_ (Table 5), so that 97.4% of the French females tested harboured _Wolbachia_. They also all harboured the same mitotype, M1. The other populations of _P. pruinosus_ tested had a

lower incidence of _Wolbachia_; the Réunion island and Tunisia populations contained about 60% infected females (Table 5). The animals from Greece, Tunisia and Réunion island that were

infected with _Wolbachia_ had the M2 mitotype, whereas the uninfected ones had the M3 mitotype (Table 4). The _Wolbachia_ transmission level was also detected by PCR in males and females

from the progenies of infected mothers (Table 5). _Wolbachia_ transmission rates are significantly different between populations (χ26=33; _P_ < 0.001). A logistic regression, comparing

France against Greece, Réunion island and Tunisia, showed significant effects of origin (Wald-test=9.85; _P_= 0.0017) and of sex (Wald-test=14.65; _P=_0.0001). Males of the Greek, Réunion

island and Tunisian populations are always uninfected by _Wolbachia_, whereas in the French population males harbour _Wolbachia_ endosymbionts at an equal rate to that of females. On

average, 90.9% of the males from French populations harboured the symbionts, and all the males were infected in Poitiers and St-Martin-du-Fouilloux (Table 5). Finally, the sequence of the

bacterial 16S rRNA genes revealed important differences between _Wolbachia_ strains from geographically distant locations. The _Wolbachia_ strain from Poitiers showed an identical sequence

to that of _P. pruinosus_ previously published, isolated from Celles sur Belle (France), a locality close to St Martin-du-Fouilloux (40 km) and Poitiers (50 km) (Bouchon et al., 1998). The

sequence of _Wolbachia_ from Réunion island exhibited differences from French symbionts (Table 6). The two sequences showed a pairwise distance of 2%, which was the highest divergence

recorded in oniscidean symbionts (Bouchon et al., 1998). Even if sequence patterns were typical of the B group of Wolbachiae (Werren & O’Neill, 1997), the high divergence recorded here

indicates that the two strains are distantly related. DISCUSSION Over the last few years, many cases of ‘species complexes’ have been described in isopods: for example, the _Orithoniscus

flavus_ complex (Dalens et al., 1996) and the _Oniscus asellus_ complex (Bilton, 1997). Even though the specimens collected in Greece, Réunion island, Tunisia and France were morphologically

indistinct and all corresponded to the _P. pruinosus_ description, it was clear that they could be separated into two groups. There were no crosses between individuals from France and those

from Réunion island, Greece and Tunisia; this reproductive isolation was precopulatory in that no mating occurred. Greek, Réunion island and Tunisian populations were interfertile and

genetically closely related. This suggested that these three populations belong to the same species. What has previously been considered to be a single species consists, in fact, of two

sibling species. These two species are geographically distinct: one is commonly found in France (‘French group’ in the following discussion) and the other one was collected in Tunisia,

Réunion island and Greece (‘southern group’ in the following discussion). These two sibling species may be included in a ‘_Porcellionides pruinosus_ species complex’, but it appears that the

taxonomic status of _P. pruinosus_ needs re-evaluation on a worldwide basis. The data from the mitochondrial DNA analysis also strengthen the distinction between two species. Using RFLP

analysis of the total mtDNA, we obtained three mitotypes of which one was strictly restricted to the French group. The two remaining mitotypes were shared in the three other populations. The

PCR-RFLP analysis of the mt 16S rDNA showed two different patterns: one is only found in the French group and the other in the southern group. The mitochondrial diversity in _P. pruinosus_,

with a considerable genetic distance (_d ≈_ 0.45) between the ‘French’ mitotype and the ‘southern’ mitotypes, suggests great divergence between the two sets of populations. Another line of

evidence confirming separated species is the nucleotide distance of 0.13 between the mitotypes, obtained with the mt 16S RNA gene portion. Tam et al. (1996) observed similar nucleotide

distance based on mt 16S rDNA between different species of the Decapoda genus _Emerita_: 0.15 between _E. analoga_ and _E. talpoida_; 0.13 between _E. analoga_ and _E. portoriciensis_; and

0.15 between _E. analoga_ and _E. benedicti_. The idea that _P. pruinosus_ has been dispersed throughout the world by the actions of man, because similar forms are observed throughout an

extensive range, is no longer acceptable. Such a spread may have occurred for the southern group which could have been transported by human activities from Greece or Africa (Tunisia) to

Réunion island, even if historical evidence for such an introduction is lacking. The dispersal of the ‘_P. pruinosus_ complex’ may be ancient and may predate the divergence of the sibling

species. We propose that the French group has been isolated from its native locality, the Mediterranean region (Vandel, 1960), and has genetically diverged from the southern group. An

isolating mechanism has evolved between these two species, so that hybridization is no longer possible between them. Additionally, the _Wolbachia_ harboured by the French and southern groups

of the _P. pruinosus_ complex are genetically different. Since both the hosts and _Wolbachia_ strains are different, the differences in feminizing pattern (presence vs. absence in males)

and in prevalence between the two associations could result from differences in coevolutionary patterns. Cross-infections are needed to discriminate which partner of the association plays a

crucial role in these differences. Nevertheless, the pattern of _Wolbachia_ infection in the southern group of the _P. pruinosus_ complex is more closely related to that of _Wolbachia_

infection in _A. vulgare_, where the males are never infected (Juchault et al., 1993). This is confirmed by the closeness of ribosomal gene sequences of _A. vulgare_ and French _P.

pruinosus_ symbionts. Variations in mitochondrial DNA are often used to trace the evolutionary history of populations (Avise et al., 1983). However, several selective forces may act on this

genetic marker, including parasite-induced selective sweeps, to confound the interpretation of data (Ballard & Kreitman, 1995). The host reproductive changes caused by _Wolbachia_

enhance the spread of the symbiont in infected populations (Werren & O’Neill, 1997). As a consequence, the infected cytoplasm increases in frequency by ‘hitchhiking’, at the expense of

uninfected cytoplasm (Turelli et al., 1992). This is very similar to the genetic hitchhiking associated with the fixation of an advantageous mutation by selection (Maynard Smith & Haigh,

1974) and will usually cause a reduction in genetic polymorphism. Its maternal inheritance and the absence of recombination makes mitochondrial DNA sensitive to such hitchhiking and a

maternally inherited factor, such as _Wolbachia_, could influence haplotype diversity (Ballard & Kreitman, 1995). Genetic hitchhiking could have caused the pattern of mtDNA variation in

_P. pruinosus_. The feminizing effect of _Wolbachia_ imparts a selective advantage to the infected females, which could have lead to a selective sweep of the associated cytoplasm, carried

passively as the microorganism spreads through the population. Populations almost entirely infected with _Wolbachia_ (French species) showed no mitochondrial polymorphism, suggesting that

the polymorphism had disappeared when invasion was complete. This hypothesis is strengthened by the presence of a unique lineage of symbionts in French populations according to the sequence

data. The only uninfected French wild-caught female shared the same mitotype with females infected by the symbiont. This could be caused by a secondary loss of _Wolbachia_ in this female. On

the other hand, populations with polymorphism for _Wolbachia_ infection (southern group) had slight mitochondrial polymorphism, and there was a link between _Wolbachia_ infection status and

mtDNA types. Here, a single mitotype was associated with the symbionts, which might be explained by a single _Wolbachia_ infection event. In these populations, uninfected individuals showed

no mtDNA polymorphism. This might be because of the small size of our samples, and/or bottlenecks experienced by populations. The general pattern of mtDNA polymorphism and _Wolbachia_

infection in _P. pruinosus_ contrasts with that found in another woodlouse: _A. vulgare_. The mtDNA in _A. vulgare_ shows considerable polymorphism, despite the presence of sex ratio

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for providing the samples from Réunion island and B. Grosset for allowing samples to be taken on his property. This study was financed by a grant from the French Ministère de l’Education

Nationale de la Recherche et de la Technologie (Acc-Sv3961098). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Université de Poitiers, Laboratoire de Génétique et Biologie des Populations de

Crustacés, UMR CNRS 6556, 40 avenue du Recteur Pineau, Poitiers, 86022, Cedex, France Isabelle Marcadé, Catherine Souty-Grosset, Didier Bouchon, Thierry Rigaud & Roland Raimond Authors *

Isabelle Marcadé View author publications You can also search for this author inPubMed Google Scholar * Catherine Souty-Grosset View author publications You can also search for this author

inPubMed Google Scholar * Didier Bouchon View author publications You can also search for this author inPubMed Google Scholar * Thierry Rigaud View author publications You can also search

for this author inPubMed Google Scholar * Roland Raimond View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Roland

Raimond. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Marcadé, I., Souty-Grosset, C., Bouchon, D. _et al._ Mitochondrial DNA variability and

_Wolbachia_ infection in two sibling woodlice species. _Heredity_ 83, 71–78 (1999). https://doi.org/10.1038/sj.hdy.6885380 Download citation * Received: 02 November 1998 * Accepted: 15 March

1999 * Published: 01 July 1999 * Issue Date: 01 July 1999 * DOI: https://doi.org/10.1038/sj.hdy.6885380 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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KEYWORDS * laboratory crosses * mitochondrial DNA polymorphism * _Porcellionides pruinosus_ * terrestrial crustacean * _Wolbachia_