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Article

Endosymbiotic Bacteria Spiroplasma and Wolbachia in a Laboratory-Reared Insect Collection

by
Roman Bykov
1,
Elena Shatalova
2,
Irina Andreeva
2,
Alevtina Khodakova
2,
Artem Ryabinin
1,
Mary Demenkova
1 and
Yury Ilinsky
1,*
1
Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia
2
Siberian Federal Scientific Centre of Agro-BioTechnologies of the Russian Academy of Sciences (SFSCA RAS), 630501 Krasnoobsk, Russia
*
Author to whom correspondence should be addressed.
Insects 2025, 16(11), 1168; https://doi.org/10.3390/insects16111168
Submission received: 2 October 2025 / Revised: 13 November 2025 / Accepted: 14 November 2025 / Published: 16 November 2025
(This article belongs to the Section Insect Behavior and Pathology)
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Simple Summary

Endosymbiotic bacteria, such as Wolbachia and Spiroplasma, can significantly influence the biology of host species. Therefore, it is essential to consider their presence and influence when working with laboratory insect cultures. We studied laboratory-reared insect stocks of non-model species for Spiroplasma and Wolbachia symbionts. Out of the thirty stocks, seven contained symbionts: five species had only Wolbachia, one had only Spiroplasma, and one carried both. We provided genotyping of the symbiont isolates and discussed the fact that laboratory non-model insects are an important source for studying host–symbiont interactions and that our findings can also be used for practical applications.

Abstract

Many insect and other arthropod species are maintained as non-model laboratory stocks and are used for fundamental and applied studies. Their biology may be affected by symbionts, such as Wolbachia and Spiroplasma. Thirty stocks of different insect species that are maintained at the Laboratory of biological control of phytophagous and phytopathogens in the Siberian Federal Scientific Centre of Agro-BioTechnologies were screened to find Spiroplasma/Wolbachia–host associations. We used 16S rDNA and fusA loci for Spiroplasma characterization and five MLST genes for Wolbachia. Seven out of thirty stocks harbored symbionts. Five stocks were infected with only Wolbachia, one with only Spiroplasma, and one with both symbionts. Two stocks were occasionally characterized by false-positive signals of Spiroplasma infection that were explained by contamination from food sources, viz. infected insects. Five Wolbachia isolates belonged to supergroup B and one to supergroup A. Only the MLST haplotype of Nabis ferus was previously known (ST-522), while the other haplotypes contained new alleles. One Spiroplasma isolate was clustered in the Ixodetis clade and another was basal to the Apis clade. We noted the importance of non-model insects for fundamental studies of host–symbiont interactions and their significance for applied research and practice.

1. Introduction

Many insect and other arthropod species are used for fundamental and applied studies. To maintain a non-model species stock, researchers collect insect specimens in the field, maintain a primary laboratory population, and characterize the population for morphology, physiology, and genetics. They also estimate it for special traits such as sex ratio, fertility, duration of development, reproductive and pre-reproductive periods, as well as the range of abiotic conditions for rearing. These traits and indicators can be affected by symbionts. Here, we have screened an insect stock collection, mainly non-model insect species, for Spiroplasma and Wolbachia symbionts. These bacteria are deeply integrated into the biology of their host species and can influence the physiological traits [1,2,3]. Characteristics of infection status and symbiont genetics of particular insect stocks can potentially be used in fundamental studies of host–symbiont interactions and their mechanisms and for applied research to optimize rearing. To share possible perspectives on such knowledge, we briefly review the biology of these symbionts and their influence on hosts.
Wolbachia are the most widespread symbiotic bacteria in arthropods, infecting more than 60% of arthropod species and some nematode species [4,5,6,7,8]. These intracellular symbionts are transmitted mostly vertically, from mother to offspring [1]. However, there is evidence of horizontal transmission of Wolbachia between host species [9,10,11]. According to phylogenetic reconstructions, there are at least 21 Wolbachia clades that are called supergroups. Strains of the A and B supergroups predominate in insect species [12,13,14,15,16,17,18,19,20,21]. Bacteria Spiroplasma are also widespread in various arthropods, infecting plants, and are even found in vertebrates including mammals [22,23]. Spiroplasma belong to the Mollicutes class, which also includes pathogens such as Mycoplasma, Entomoplasma, Phytoplasma, and others. There are two modes of spiroplasmas transmission in hosts: vertical (from mother to offspring) and horizontal (via common substrates) [2]. More than 40 species of Spiroplasma are described and they are subdivided into several phylogenetic clades: Apis, Citri–Chrysopicola–Mirum (CCM) and Ixodetis [24,25]. The latter is the most widespread in arthropods [26].
Both Wolbachia and Spiroplasma may have a wide range of effects on their hosts. In particular, both bacteria induce male killing and cytoplasmic incompatibility in different insect species [27,28,29,30,31,32,33,34]. Wolbachia also induce feminization and thelytokous parthenogenesis [32]. Besides these reproductive abnormalities, some mutualistic effects of the symbionts, such as suppression of mutations, increased fitness and longevity for Wolbachia [35,36,37,38], and protection from parasitoids, nematodes, and fungi for both bacteria [39,40,41,42] are reported. Some Spiroplasma species are pathogens of plants, arthropods, and vertebrates [43,44,45]. Co-infection with Wolbachia and Spiroplasma is shown for a spider mite Tetranychus truncatus [46,47], where the symbionts alter the expression of many genes, increase female fecundity and male hatchability, and accelerate development, but also induce cytoplasmic incompatibility [46]. In addition, co-infection alters host plant defense, which is manifested in decreased expression of jasmonic and salicylic acid responsive genes [48].
Current questions in studies of these bacteria are regarding their effects on hosts and the possibility of their use for practical purposes, e.g., for increasing host fecundity and defending it from environmental factors. Another question concerns the mechanisms of the symbiont–host interaction based on new model systems. In the present study, we aim to find potential new models of Spiroplasma/Wolbachia–host associations that can be used for fundamental and applied research.

2. Material and Methods

2.1. Insect Collection

Thirty insect stocks were used in this study (Table 1). All stocks were maintained at the Laboratory of biological control of phytophagous and phytopathogens SFSCA RAS, Krasnoobsk, Russia (collection supported by the FNUU-2024-0002). The origin of most stocks was field collections in the forest–steppe zone of Western Siberia. Insects were collected using standard entomological methods. Some stocks were established based on the material provided by other researchers (private stocks). The Macrolophus pygmaeus stock was founded from specimens collected in greenhouse farming in Novosibirsk Province (Russia). All stocks were maintained independently without crosses with any other stocks. Species identification was performed according to morphological traits using keys in the relevant guides [49,50,51,52]. The stocks were kept in plastic boxes (30 cm × 40 cm × 25 cm) or in cages (40 cm × 40 cm × 40 cm) covered with tulle. Environmental conditions for insect maintenance were as follows: temperature range of 24–27 °C, humidity of 30–60%, and lighting for no less than 12 h per day. The insects were fed 2–3 times per week with the appropriate nutrient substrate (Table 1) [53,54,55].

2.2. DNA Extraction

All insect specimens were frozen at −20 °C prior to DNA extraction. Depending on specimen size, we used part of an insect, a whole insect, or a pool of insects. In particular, for insects larger than 15 mm (Acheta domesticus, Arma custos, G. bimaculatus, L. oleracea, Platymeris biguttatus, Psytalla horrida) and for all Coccinellidae, an abdomen or gut and reproductive tissues were used because the reproductive and digestive systems were the focus of the Wolbachia and Spiroplasma analysis (Table S1). The whole insect was used in the case of specimens in the range of 5–15 mm (B. rufimanus, C. formosa, M. pygmaeus, Nabis species, Nesidiocoris tenuis, P. maculiventris). A pool of 5–10 specimens of insects smaller than 5 mm was used for DNA extraction (Trissolcus kozlovi, Trialeurodes vaporariorum, aphids). DNA extraction was repeated from different generations of stocks. In addition to extraction of adult specimens, we also used eggs, larvae, and nymphs in some cases. Material for extraction was homogenized in 200–400 µL of extraction buffer (10 mM Tris-HCl at pH 8.0, 25 mM EDTA, 0.5% of SDS, and 0.1 M NaCl) depending on specimen size and incubated at 56 °C for 1–2 h. After precipitation, DNA was dissolved in 100–200 µL deionized water. The quality of DNA samples was estimated using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by amplification with primer set LCO1490/HCO2198 [56].

2.3. PCR and Sequencing

For Wolbachia screening, we used two loci; in particular, we applied a nested PCR approach for the ftsZ gene and conventional PCR for the coxA gene. In the first round of nested PCR, the primer set ftsZunif/ftsZunir [14] was used, and in the second round, ftsZF1/ftsZR1 primers [57] were used. For the coxA gene, the primer set coxAF1/R1 [57] was used. The Bi90 strain of Drosophila melanogaster [58] was used as a positive control for Wolbachia infection and sterile water as a negative control. Screening for Spiroplasma infection was performed using the primer set SpiF1/SpiR3 [59] for the 16S rDNA locus (fragment size of approximately 1067 bp). An infected sample of Tabanidae sp. was used as a positive control and sterile water as a negative control. Wolbachia-positive samples were characterized using five loci of the MLST protocol [57]. Spiroplasma-positive samples were sequenced using the above-mentioned 16S rDNA primers. In addition, we elaborated a primer set for the elongation factor G (fusA) gene. According to our analysis, fusA is a core gene of Spiroplasma genomes with relatively conserved regions, which allowed us to design the following oligonucleotides: SfusAF 5′-CACGTTGAYTTYACWGTTGAAGT-3′ and SfusAR 5′-CCAACAAAWGGGTCWGTCAT-3′. This primer set produces an approximately 700 bp amplicon. Infected insect stocks were characterized using the barcoding region of the co1 gene with primer set LCO1490/HCO2190.
PCR cycling conditions were 95 °C for 5 min, followed by 35 cycles at 95 °C for 15 s for co1 and 16S rDNA and 10 s for fusA, 53 °C for 1 min for co1, 40 s for 16S rDNA, 55 °C for 30 s for fusA, 72 °C for 3 min for co1, 50 s for 16S rDNA and fusA, plus a final elongation step of 72 °C for 3 min for co1 and 2 min for 16S rDNA and fusA. For nested PCR of MLST genes, cycling conditions were as follows: first round: 95 °C for 5 min, followed by 15 cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 40 s; second round: 95 °C for 5 min, followed by 30 cycles at 95 °C for 5 min, 55 °C for 30 s, and 72 °C for 30 s. PCR products were purified with exonuclease I from E. coli and sequenced using the BrilliantDye Terminator Cycle Sequencing Kit (Nimagen, Nijmegen, The Netherlands) with the same primers as mentioned for PCR. All sequences were deposited in the GenBank database under accession numbers PX259646-PX259652 for co1; PX257974-PX257975 for Spiroplasma 16S rDNA gene; PX273486-PX273487 for Spiroplasma fusA gene; and PX273488-PX273493, PX273494-PX273499, PX273500-PX273505, PX273506-PX273511, and PX273512-PX273517 for Wolbachia gatB, coxA, hcpA, ftsZ, and fbpA, respectively (Table S2).

2.4. Phylogenetic Analysis

Analysis and assembly of sequences were performed using MEGA12 v.12.0.10 software [60]. All co1 sequences were checked in the GenBank [https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 1 September 2025)] and BOLD [https://id.boldsystems.org/ (accessed on 1 September 2025)] databases. Sequences of MLST Wolbachia loci were checked in the PubMLST [61] database. New Wolbachia alleles were additionally checked in the GenBank database. Sequences of Spiroplasma 16S rDNA and fusA loci were checked in the GenBank database to find the most similar isolates. To reconstruct the phylogenetic relationships of Wolbachia isolates, the sequences of MLST genes were concatenated in the order gatB-coxA-hcpA-ftsZ-fbpA and aligned in MEGA12 using the Muscle algorithm [62]. Additional sequences of the closest isolates were included in the phylogenetic analysis. The total alignment length was 2079 bp. The Kimura two-parameter model of nucleotide substitutions with Gamma distribution (K2G) was chosen as the best fit in MEGA12. For Spiroplasma 16S rDNA and fusA loci, all sequences, including additional sequences of the closest isolates from the GenBank database, were aligned in MEGA12 using the Muscle algorithm and then trimmed to the shortest sequence. Total alignment lengths were 1011 and 626 bp, respectively. The best-fit model for phylogenetic reconstruction was K2G for 16S rDNA and General Time Reversible with Gamma distribution for fusA. Phylogenetic trees were reconstructed using the Maximum Likelihood (ML) algorithm with 1000 bootstrap iterations.

3. Results

Screening for Wolbachia and Spiroplasma infection revealed seven out of thirty insect stocks that stably harbored symbionts (Table 1). Five stocks were infected with only Wolbachia, one stock with only Spiroplasma, and the Nabis sp. stock harbored both symbionts. Evidence of stable persistence of bacteria was obtained by checking infection status in different insect generations and different insect tissues, and through genetic characterization of each symbiont isolate. In the case of T. molitor, we noted that the prevalence of Spiroplasma infection varied; not all specimens were infected. Two insect stocks were occasionally characterized by positive signals of Spiroplasma infection; however, this result was explained by contamination from the food source. In particular, Spiroplasma infection in A. custos and P. maculiventris stocks could be found only after feeding with T. molitor that was truly infected. Moreover, the sequences of 16S rDNA Spiroplasma isolates from A. custos and P. maculiventris were identical to the bacterial isolate of T. molitor. We could not determine whether Spiroplasma-positive signals in these two stocks reflected only a trace of symbiont DNA or whether Spiroplasma temporarily inhabited these species.
Symbiont-harboring stocks were characterized using the barcoding region of the host mitochondrial co1 gene (Table 2). Morphological and co1 barcoding identification of T. molitor, M. pygmaeus, and N. ferus (collection of 2024) were in agreement. However, for M. pygmaeus, the complete identity of the barcoding region was also found with M. caliginosus in both databases. Two stocks of Chrysopa were identified by co1 barcoding as C. formosa, and their sequences differed by two substitutions. The stock of Nabis sp. was not identified because several species were closely related by the barcoding sequence. The number of singletons between “Nabis sp. (2022)” and “Nabis ferus (2024)” was 74 (89% identity). The stock of parasitoid wasp, which was identified by Dr. A. Timokhov as Trissolcus kozlovi, showed no similarity with determined species in the BOLD database but had high similarity with T. kozlovi in the GenBank database.
Five out of six Wolbachia isolates belong to supergroup B according to phylogenetic analysis of concatenated MLST genes, and only the isolate of T. kozlovi belongs to supergroup A (Figure 1). Only the MLST haplotype of N. ferus was previously known (ST-522), while other haplotypes contained new alleles according to both the PubMLST and GenBank databases, as well as new combinations of known alleles. The haplotype of M. pygmaeus was very close to ST-431 (one mismatch), which was previously found in the parasitoid wasp Encarsia inaron (PubMLST id-1676 and -1677). Wolbachia haplotypes of C. formosa stocks were identical, and haplotypes of Nabis stocks were closely related.
Two isolates of Spiroplasma infection were genotyped for 16S rDNA and fusA loci. Reconstruction of phylogenetic relationships revealed that Spiroplasma of Nabis sp. clustered in the Ixodetis clade, whereas Spiroplasma of T. molitor was basal to the Apis clade of Spiroplasma (Figure 2 and Figure 3). According to the GenBank database, the sequence of 16S rDNA Spiroplasma isolated from T. molitor was 99.81% identical to two sequences designated as uncultured bacterium clones (accession numbers DQ163945 and DQ163951), which were also isolated from T. molitor [63]. The closest determined Spiroplasma was S. taiwanense (NR_121701) with 95.72% identity. For the fusA gene, the closest isolate was S. chinense (CP043026) with 85.27% identity.

4. Discussion

4.1. Barcoding Identification of the Symbiont-Harbored Species

Since Wolbachia and, in some cases, Spiroplasma are maternally inherited bacteria, we analyzed the co1 barcode region for host strains that were found to be infected. In addition to host mtDNA association with symbionts, we were able to compare morphological and genetic identification of the species, and, in most cases, these results were in agreement (Table 2). For Macrolophus pygmaeus, there were two species matches: M. pygmaeus and M. caliginosus (syn. M. melanotoma). According to molecular data, they are different species; however, they are hardly distinguishable by morphology [64,65]. The co1 sequences of M. caliginosus that are identical to our data on M. pygmaeus were obtained in a study where the authors noted, “The specimens of M. caliginosus and M. pygmaeus that we received from our sources could not be diagnostically resolved as all samples showed identical sequences” [66]. Therefore, we assume that our stock is M. pygmaeus.

4.2. Symbionts in Laboratory Stocks

We found seven laboratory insect stocks that were stably infected with Spiroplasma and/or Wolbachia bacterial symbionts. Among the studied species, Spiroplasma infection was noted in T. molitor based on microbiome studies [67,68]; however, there was no particular consideration of Spiroplasma. Comparison of the 16S rDNA sequences of Spiroplasma isolates from our and previous reports indicates the same origin of the infection. In the Nabidae family, Spiroplasma infection has not been reported previously; thus, our study is the first case of the Nabis–Spiroplasma symbiotic association.
Regarding Wolbachia infection, only Macrolophus pygmaeus was previously reported to be infected [69]. Moreover, it was shown that Wolbachia induced strong cytoplasmic incompatibility, and it was demonstrated how the infection was distributed in the bug’s organs. In particular, the symbionts were found in salivary glands and ovaries but not in the gut. Other cases of host–Wolbachia associations found in our study are new; however, some facts about Wolbachia are known in closely related species. For instance, Wolbachia infection was reported for Chrysopa lacewings [id1163 in the PubMLST database], Nabis bugs [70,71], and different Trissolcus species [72].
According to previous papers, we could expect to find Wolbachia or Spiroplasma in some other species studied here. Both symbionts were found in two-spot ladybird (Adalia bipunctata) [28,73,74,75,76,77,78] and harlequin ladybird (Harmonia axyridis) [75,79,80,81], as well as in the bird cherry-oat aphid (Rhopalosiphum padi) [82,83], the black bean aphid (Aphis fabae) [20,21,84,85], and in the tomato bug (Nesidiocoris tenuis) [86,87]. Wolbachia was found in a laboratory stock of house cricket (Acheta domesticus) [88] and in the seven-spot ladybird (Coccinella septempunctata) [89], the glasshouse whitefly (Trialeurodes vaporariorum) [90,91,92,93], the diamondback moth (Plutella xylostella) [94,95,96], and the cabbage aphid (Brevicoryne brassicae) [97]. Spiroplasma was reported in Zophobas morio [98] and the white-eyed assassin bug (Platymeris biguttatus) [71].

4.3. Genetics of the Symbionts

Wolbachia variants isolated here belong to supergroups A and B, which are widespread in insects. Only Wolbachia MLST haplotype ST-522 found in N. ferus (2024) was previously reported in the PubMLST database. Unfortunately, we do not know whether it was found in the same host or not because there are no details on the isolate in the database. Other Wolbachia variants in our study contained new alleles or new combination of known alleles. This is the first report of a complete MLST profile of Neuroptera species; two Wolbachia isolates of Chrysopa formosa have an identical MLST haplotype. Closely related Wolbachia variants are found in different insect orders. Previously, a closely related allele of the fbpA locus (one substitution) was represented in the PubMLST and GenBank databases (id-1163, KX843371), and also for Wolbachia of the Chrysopa genus but from Polynesia [99]. This is the first complete MLST profile obtained for a representative of a Trissolcus host. Earlier, the diversity of Wolbachia in five Trissolcus species was characterized by the wsp gene [72]. The authors observed similar variants that clustered into supergroup B. In contrast, the isolate of T. kozlovi belonged to supergroup A of Wolbachia. The haplotype of M. pygmaeus contained previously known alleles but in a new combination. The most similar haplotype was found in parasitoid wasp Encarsia inaron, which is used in biocontrol against whiteflies. E. inaron and M. pygmaeus could share the same whiteflies as food substrate, and therefore this could be an arena of Wolbachia exchange between predator species. Such a pattern of Wolbachia horizontal transmission was presented by Ahmed et al. [11]. In the case of M. pygmaeus, the symbiont Wolbachia was present in salivary glands [69], and feeding by this predator occurred by injecting saliva into prey and sucking out digestive products. This process can be interrupted at the saliva injection stage, and the prey stays alive but has Wolbachia bacteria from Macrolophus. Also, the predator could hypothetically acquire new Wolbachia from prey at the sucking out stage. Of course, such speculation should be tested in experiments. Here, we note that Machtelinckx et al. [69] indicated that “Performing MLST may provide more evidence on the phylogenetic relationship between the Wolbachia strains in the whitefly and the mirid bug”. We compared Wolbachia haplotypes of Bemisia tabaci from the PubMLST database [https://pubmlst.org/bigsdb?db=pubmlst_wolbachia_isolates (accessed on 1 September 2025)] with our isolate of M. pygmaeus and found no similarities (Figure 4). However, it is obvious that the genetic diversity of Wolbachia in mirid bugs and whiteflies was underestimated. For instance, a number of Wolbachia variants were reported only for one species, Bemisia tabaci (Figure 4). M. pygmaeus also had different Wolbachia variants. Machtelinckx et al. [69] sequenced one out of five MLST loci (fbpA), and it drastically differed from the allele found in our isolate. Therefore, the finding of the same variants in prey and predator cannot be ruled out; however, direct evidence of such symbiont transmission should be obtained in an experiment.
Two Spiroplasma isolates found here are distantly related according to conserved genetic markers. Spiroplasma of Nabis sp. belongs to the Ixodetis clade, the most widespread in arthropods [26], while the isolate of T. molitor is a unique branch that is basal to the Apis clade. The sequences of our and previously reported isolates of T. molitor are identical (Figure 2), which indicates broad distribution of this infection in T. molitor stocks. It is known that spiroplasmas of the Ixodetis clade can have different effects on hosts [2]. They can increase hatchability and decrease development time [47] and defend against parasitoids and fungal pathogens [100,101], but can also induce male killing [30,33,102,103,104] and, moreover, can be pathogenic for hosts [105]. Therefore, some of these effects can be expected in Nabis sp. There are no data on any Spiroplasma effects on T. molitor biology, and the unique placement of the isolate found in our study only increases interest in studying this symbiotic association.
We should note that there are two cautions in screening for Wolbachia and Spiroplasma, especially in non-model species. First is the risk of obtaining false-positive signals due to transient microbiota from infected substrates and the frequent presence of “weed” mites (e.g., Tyrophagous spp.) in insect stocks. Second is the risk of obtaining false-negative signals due to variation in infection prevalence in insect stocks, which could be a result of the imperfect transmission of infection [33,106] and low bacterial density in individuals or in some organs and tissues [107,108].

4.4. How These Symbionts Can Be Used in Biocontrol

In the present study, we firstly reported on Wolbachia and Spiroplasma in species with potential for biocontrol. Specifically, Wolbachia and Spiroplasma were detected in the predatory bug Nabis ferus. Additionally, Wolbachia was identified for the first time in Chrysopa formosa and Trissolcus kozlovi. All but one of the Wolbachia isolates were characterized by novel MLST alleles or novel combinations of previously known alleles. Also, we provide the first genetic characterization of Spiroplasma isolated from Tenebrio molitor. These findings pave the way for further investigation into the roles of these symbionts in their hosts’ biology.
The application of Wolbachia and Spiroplasma in agriculture is at the initial stages; there are as yet no use-ready technologies for these symbionts in applied science. However, several promising directions for their utilization can be identified. Notably, Wolbachia strains from supergroup B found in Trichogramma wasps, widely used agents in biological pest control, are known to induce parthenogenesis, resulting in the production of all-female offspring (thelytoky) [109]. By transferring the thelytokous Wolbachia strains to bisexual Trichogramma stocks, it is possible to create female-only lines, which can significantly lower the cost of mass wasp production and enhance the efficiency of biocontrol programs [110]. Furthermore, certain Wolbachia strains possess genes conferring nutritional independence, such as the biotin operon [111]. The introduction of such strains into biocontrol species could improve the sustainability and management of production stocks. The defensive role of Spiroplasma [2] could be used for applied species to protect them from natural enemies [112,113,114] or even from abiotic factors [115]. In summary, while technologies utilizing Wolbachia and Spiroplasma are not yet available, active research supports their potential for improving efficiency, cost-effectiveness, and environmental resilience in agriculture.

5. Conclusions

Some of the symbiont-harboring species considered here are maintained in laboratory stocks worldwide, which means that other researchers can find Wolbachia and Spiroplasma in their own stocks. Our other original stocks that also harbor symbionts can be successfully established and maintained elsewhere because they are easily cultivated under laboratory conditions. We also list species that are cultivated and could be infected according to previous reports. All of them are potential sources for fundamental studies of symbiont–host interactions, as well as for applied research to improve productivity, cultivation methods, and other aspects of practical insect use.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/insects16111168/s1. Table S1: Life stages and DNA extraction sources; Table S2: Number of specimens examined and sequenced for each stock.

Author Contributions

Conceptualization, R.B. and Y.I.; methodology, R.B., E.S., I.A., A.K., A.R., M.D., and Y.I.; validation, R.B., E.S., A.R., M.D., and Y.I.; formal analysis, R.B. and Y.I.; investigation, E.S., R.B., A.R., M.D., and Y.I.; resources, I.A. and Y.I.; data curation, R.B., E.S., and Y.I.; writing—original draft preparation, R.B. and Y.I.; writing—review and editing, R.B., E.S., and Y.I.; visualization, R.B.; supervision, Y.I.; project administration, Y.I.; funding acquisition, Y.I. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by RSF No. 24-24-00378.

Data Availability Statement

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

Acknowledgments

We are grateful to A. Timokhov (Moscow State University) for consultation, to D. Fedorov for technical support and to the FWNR-2022-0019 for Wolbachia screening support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The ML phylogenetic tree of Wolbachia isolates based on five concatenated MLST genes (gatB-coxA-hcpA-ftsZ-fbpA) reconstructed with model K2p + G and 1000 bootstrap iterations. Wolbachia sequence types (STs) and/or host species are provided; isolates of this study are in bold. ST-1 and -19, -9 and -41, and -8 were chosen as supergroups A, B, and F references, respectively. Bootstrap values of 75 and higher are indicated.
Figure 1. The ML phylogenetic tree of Wolbachia isolates based on five concatenated MLST genes (gatB-coxA-hcpA-ftsZ-fbpA) reconstructed with model K2p + G and 1000 bootstrap iterations. Wolbachia sequence types (STs) and/or host species are provided; isolates of this study are in bold. ST-1 and -19, -9 and -41, and -8 were chosen as supergroups A, B, and F references, respectively. Bootstrap values of 75 and higher are indicated.
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Figure 2. The ML phylogenetic tree of Spiroplasma isolates based on 16S rDNA locus reconstructed with model K2p + G and 1000 bootstrap iterations. GenBank accession numbers and Spiroplasma species are provided; isolates of this study are in bold. Bootstrap values of 75 and higher are indicated.
Figure 2. The ML phylogenetic tree of Spiroplasma isolates based on 16S rDNA locus reconstructed with model K2p + G and 1000 bootstrap iterations. GenBank accession numbers and Spiroplasma species are provided; isolates of this study are in bold. Bootstrap values of 75 and higher are indicated.
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Figure 3. The ML phylogenetic tree of Spiroplasma isolates based on fusA locus reconstructed with model GTR + G and 1000 bootstrap iterations. GenBank accession numbers and Spiroplasma species are provided; isolates of this study are in bold. Bootstrap values of 75 and higher are indicated.
Figure 3. The ML phylogenetic tree of Spiroplasma isolates based on fusA locus reconstructed with model GTR + G and 1000 bootstrap iterations. GenBank accession numbers and Spiroplasma species are provided; isolates of this study are in bold. Bootstrap values of 75 and higher are indicated.
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Figure 4. The ML phylogenetic tree of Wolbachia haplotypes from Macrolophus pygmaeus from our study (in bold) and Bemisia species from the PubMLST database based on concatenated MLST sequences. Reconstructed with model T2p + G and 1000 bootstrap iterations. PubMLST id numbers, host species, and Wolbachia sequence types (STs) are provided. Bootstrap values of 75 and higher are indicated.
Figure 4. The ML phylogenetic tree of Wolbachia haplotypes from Macrolophus pygmaeus from our study (in bold) and Bemisia species from the PubMLST database based on concatenated MLST sequences. Reconstructed with model T2p + G and 1000 bootstrap iterations. PubMLST id numbers, host species, and Wolbachia sequence types (STs) are provided. Bootstrap values of 75 and higher are indicated.
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Table 1. List of species maintained in the Laboratory and their characteristics: purpose of use, origin, nutrient substrate, and infection status.
Table 1. List of species maintained in the Laboratory and their characteristics: purpose of use, origin, nutrient substrate, and infection status.
OrderSpeciesPurpose of CultureOrigin (Year)Nutrient
Substrate		WolbachiaSpiroplasma
OrthopteraAcheta domesticusIFPS, Msk, (2022)Vegetables00
ColeopteraAdalia bipunctataBCSFSCA, Nsk (2024)Aphids00
HemipteraAphis fabaetest, IFSFSCA, Nsk (2017)Bean00
HemipteraArma custosBCSFSCA, Nsk (2023)Tenebrio molitor00*
HemipteraBrevicoryne brassicaetest, IFSFSCA, Nsk (2023)Cabbage00
ColeopteraBruchus rufimanustestPS, Msk (2024)Mash00
NeuropteraChrysopa formosa 1BCSFSCA, Nsk (2018)Aphids10
NeuropteraChrysopa formosa 2BCSFSCA, Nsk, (2024)Aphids10
ColeopteraCoccinella septempunctataBCSFSCA, Nsk (2022)Aphids00
OrthopteraGrillus bimaculatusIFPS, Msk (2022)Vegetables00
ColeopteraHarmonia axyridisBCSFSCA, Nsk (2022)Aphids00
ColeopteraHarmonia (Leis) dimidiataBCVIZR, SPb (2024)Aphids00
ColeopteraHippodamia variegataBCSFSCA, Nsk (2022)Aphids00
LepidopteraLacanobia oleraceatest, IFSFSCA, Nsk (2023)Cabbage00
HemipteraMacrolophus pygmaeusBCCS (2017)T. vaporariorum10
HemipteraMegoura viciaetest, IFSFSCA, Nsk (2017)Bean00
HemipteraNabis sp. BCSFSCA, Nsk (2023)Aphids11
HemipteraNabis ferusBCSFSCA, Nsk (2024)Aphids10
HemipteraNesidiocoris tenuisBCVIZR, SPb (2023)T. vaporariorum00
HemipteraPlatymeris biguttatustestPS, T (2023)T. molitor, Z. morio00
LepidopteraPlutella xylostellaIFSFSCA, Nsk (2019)Rape00
HemipteraPodisus maculiventrisBCVIZR (2017)T. molitor, Z. morio00*
HemipteraPsyttala horridatestPS, K (2024)T. molitor, Z. morio00
HemipteraRhopalosiphum paditest, IFSFSCA, Nsk (2018)Wheat00
HemipteraSchizaphis graminumtest, IFSFSCA, Nsk (2017)Wheat00
ColeopteraSubcoccinella vigintiquatuorpunctatatestSFSCA, Nsk (2023)Alfalfa00
ColeopteraTenebrio molitortest, IFPS, Nsk (2020)Bran, vegetables01
HemipteraTrialeurodes vaporariorumtest, IFSFSCA, Nsk (2021)Tobacco00
HymenopteraTrissolcus kozloviBCSFSCA, Nsk (2021)P. maculiventris10
ColeopteraZophobas morioIFPS, Nsk (2020)Bran, vegetables00
Footnotes: IF—insect food, BC—biological control, test—for experimental procedures, SFSCA—Siberian Federal Research Centre of Agro-BioTechnologies of the Russian Academy of Sciences, VIZR—All-Russian Institute of Plant Protection, PS—private stock, CS—commercial stock, SPb—Saint Petersburg, Nsk—Novosibirsk Province, Msk—Moscow city, K—Kaluga city, T—Tolyatti, 0*—uninfected but occasionally false-positive signal due to contamination by a food substrate.
Table 2. Morphological and barcoding identification of Spiroplasma-/Wolbachia-infected species.
Table 2. Morphological and barcoding identification of Spiroplasma-/Wolbachia-infected species.
Morphological Identification (GenBank Acc. Num.)BOLD (% Identity)GenBank (% Identity)
Trissolcus kozlovi (PX259647)Scelionidae (99.53)T. kozlovi (99.12)
Tenebrio molitor (PX259651)T. molitor (99.85)T. molitor (99.85)
Nabis sp. (PX259649)N. rugosus, N. pseudoferus, N. brevis (99.8), N. ericetorum (99.7), N. ferus (99.54)N. rugosus, N. pseudoferus (99.7), N. ferus (99.54)
Nabis ferus (PX259650)N. ferus (99.7)N. ferus (99.54)
Chrysopa formosa 1 (PX259648)C. formosa (100)C. formosa (100)
Chrysopa formosa 2 (PX259652)C. formosa (99.85)C. formosa (99.7)
Macrolophus pygmaeus (PX259646)M. pygmaeus, M. caliginosus (100)M. pygmaeus, M. caliginosus (100)
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Bykov, R.; Shatalova, E.; Andreeva, I.; Khodakova, A.; Ryabinin, A.; Demenkova, M.; Ilinsky, Y. Endosymbiotic Bacteria Spiroplasma and Wolbachia in a Laboratory-Reared Insect Collection. Insects 2025, 16, 1168. https://doi.org/10.3390/insects16111168

AMA Style

Bykov R, Shatalova E, Andreeva I, Khodakova A, Ryabinin A, Demenkova M, Ilinsky Y. Endosymbiotic Bacteria Spiroplasma and Wolbachia in a Laboratory-Reared Insect Collection. Insects. 2025; 16(11):1168. https://doi.org/10.3390/insects16111168

Chicago/Turabian Style

Bykov, Roman, Elena Shatalova, Irina Andreeva, Alevtina Khodakova, Artem Ryabinin, Mary Demenkova, and Yury Ilinsky. 2025. "Endosymbiotic Bacteria Spiroplasma and Wolbachia in a Laboratory-Reared Insect Collection" Insects 16, no. 11: 1168. https://doi.org/10.3390/insects16111168

APA Style

Bykov, R., Shatalova, E., Andreeva, I., Khodakova, A., Ryabinin, A., Demenkova, M., & Ilinsky, Y. (2025). Endosymbiotic Bacteria Spiroplasma and Wolbachia in a Laboratory-Reared Insect Collection. Insects, 16(11), 1168. https://doi.org/10.3390/insects16111168

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