https://orcid.org/0000-0002-4465-1169
Figures
Abstract
This study focuses on the cowpea weevil, Callosobruchus maculatus, a globally distributed grain pest that affects cereals and pulses. Using chemicals to store grains can harm pest control and pose risks to consumers and the environment. The facultative intracellular symbiont bacteria Wolbachia can affect host’s reproductive capacities in a variety of ways, which makes it useful in the management of pests such as C. maculatus. The main goal of the study was to identify Wolbachia diversity in the C. maculatus population. Phylogenetic analysis utilized mitochondrial COI and 12S rRNA genes to identify the host C. maculatus, while screening for Wolbachia was conducted using genes (wsp, coxA, and ftsZ) genes. Molecular phylogenetic analysis of the Wolbachia genes resulted in one new Wolbachia strain (wCmac1) in C. maculatus populations and contrasting already published data of other Callosobruchus strains. The study discussed the detection of Wolbachia and its phylogenetic comparison with other C. maculatus and Coleopteran populations. It is important to take these findings into account when considering host-pathogen interactions.
Citation: Rasool B, Younis T, Zafar S, Parvaiz A, Javed Z, Rasool I, et al. (2024) Incidence of endosymbiont bacteria Wolbachia in cowpea weevil Callosobruchus maculatus Fabricius (Coleoptera, Chrysomelidae). PLoS ONE 19(12): e0313449. https://doi.org/10.1371/journal.pone.0313449
Editor: Nafiu Bala Sanda, Bayero University Kano, NIGERIA
Received: March 17, 2024; Accepted: October 23, 2024; Published: December 10, 2024
Copyright: © 2024 Rasool et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The manuscript's figures, tables, and supplemental materials contain all the data. Sequences are submitted in the GenBank, and accession numbers are mentioned in MS. Some sequences will be available after acceptance of MS.
Funding: This research is partially supported for the purchase of molecular biology reagents used in the experiments by the Higher Education Commission of Pakistan (HEC) project.
Competing interests: NO authors have competing interests.
1. Introduction
Pulses are more affordable source of protein for many people around the world, particularly in developing countries [1]. Because some varieties contain even more protein, they are significant and essential in terms of nutrition [2]. Pulses are recognized for their effective and multiple participatory roles in enhancing environmental and agricultural sustainability [1, 3].
The potential pest control quality, quantity, and actual benefits of pulse pests for meeting global and national targets are essential to supporting policy development and integrated pest control planning. Priorities for global strategic research on pulse crops include end-user needs, transformative potential, and sustainability. To effectively control pests, research is required to develop a clear understanding of pest management, environmentally friendly solutions, and socioeconomic benefits [4].
Insects comprised above seventy percent of species of arthropods and Coleopterans [5]. According to estimation, about 70 to 95% of all beetle species remain undescribed [6]. Beetles are the model organisms often used in biological, medical, and environmental research [7]. Among the stored grain pests, the bruchids are essential species in pulses during storage [8]. Cowpea weevil Callosobruchus maculatus F. (Coleoptera: Chrysomelidae: Bruchinae) has worldwide distribution except for Antarctica. This agricultural pest ranges throughout the tropical and subtropical world [9–11].
Callosobruchus maculatus is one of the important factors for economic loss to the pulses and a substantial pest of stored grains [8, 12–14]. It is a model organism in population biology and affects all types of pulse fields. This pest often originates in pulse debris, may build up huge populations, and has emerged as the principal post-harvest pest. Moreover, this pest multiplies rapidly within a short time and can cause quantitative and qualitative losses of up to 100% of stored pulses [8, 14–16].
The utilization of synthetic chemicals is a common practice to control pests on pulses during storage [17]. These strategies led to several health risks, such as residual toxicity, ecological contamination, natural imbalances, pollution of the environment, insect resistance, secondary insect epidemics, phytotoxicity, chemical residues in food, and disruption of biological fauna [15, 17–19]. Consequently, there is a need to integrate advanced biotechnological tools to control this pest.
Endosymbiotic microorganism associations are quite pervasive in insects and arthropods [20]. There is diversified interdependency present during the interactions of the host and endosymbiotic relationships [21]. Wolbachia alpha-proteobacteria, detected in many species of arthropods, can transmit maternally. Wolbachia has been detected in 10–70% of examined hosts [21–22]. Wolbachia in Coleopteran species is well established and documented [23].
It has gotten attention due to its vast occurrence and possible applications in pest and disease management. Wolbachia is well known for causing reproductive changes in host tissues through mechanisms such as parthenogenesis, feminization, male killing, and cytoplasmic incompatibility (CI) [24]. Cytoplasmic incompatibility (CI) occurs when a bacterium causes sterility in crosses between Wolbachia-infected males and wild females. This can be used to control insect pests through the incompatible insect technique (IIT). Releasing only males is crucial for a successful IIT-based approach. Previous research has shown positive effects of IIT on insect disease vectors and agricultural pests. Wolbachia-based IIT could be an effective and environmentally friendly pest management approach [25–28]. Consequently, Wolbachia is regarded as a selfish genetic element for its engagement with the host in an evolutionary sense [24], making it an attractive subject of evolutionary and molecular biology [24, 29]. Therefore, if the endosymbiotic microorganism prevails in C. maculatus, it will offer us a good configuration to explore the host-symbiont dynamics [23, 30].
For population genetic and phylogenetic research in a variety of organisms, insect mtDNA, which is inherited maternally, is an invaluable resource [31–33]. The advent of polymerase chain reaction PCR has made primers for mitochondrial genes amplification accessible [32, 34]. Mitochondrial assessment for the characterization and identification of different arthropod species is frequently used. For insects and related groups, 12S rRNA fragments are universal. For the identification of insect species, the cytochrome c oxidase subunit I (COI) DNA barcode region is thought to be a useful and reliable diagnostic tool because of its fast mutation rate to differentiate closely related species [35]. Wolbachia outer surface protein (wsp) is considered to be the valid markers for the exploration of endosymbiotic bacteria [36]. In comparision, multilocus strain typing (MLST) is an appropriate and fast detectable approach that is recognized for exploring bacterial strains, including Wolbachia. MLST exploits five conserved housekeeping genes, including coxA, gatB, ftsZ, hcpA and fbpA [37, 38]. This approach is a promising tactic for exploring and cataloging strains and studying the molecular ecology, biodiversity, and development of Wolbachia [39–41]. There are currently 17 Wolbachia supergroups known to exist and identified as A to R [24, 42, 43]. Focused research objectives on Wolbachia had great potential for possible application in agricultural pest management and vector borne diseases. The application of chemical control used for the control of C. maculatus [44, 45] jeopardized the resistant populations, human health, and the environment [45]. There were already numerous insect induced pest incursions of stored grains in the storage area. Controlling stored product insects primarily aims to eradicate these residual populations. Ecologically risky methods of controlling bruchids are not very suitable for small-scale, economically viable farmers [46]. Wolbachia manipulates its host’s biology through phenotypic alterations, including cytoplasmic incompatibility [24]. This leads to the incompatible insect technique (IIT), which effectively controls insect disease vectors and agricultural pests [25, 26, 28]. The current study set out to look into Wolbachia infection in the various natural populations of C. maculatus. Nevetheless, molecular identification of C. maculatus through mitochondrial (COI) and 12S rRNA genes was accomplished. Phylogenetic analysis was performed using marker gene sequences. Wolbachia strains were screened to test the hypothesis that advanced typing (wsp, coxA, and ftsZ) genes in addition to (12S rRNA, COI) of the C. maculatus populations. This investigation represents the first step toward a longer-term objective of determining whether Wolbachia-induced strains can be used as a novel, eco-friendly tool for C. maculatus management.
2. Materials and methods
The present research was conducted to explore the endosymbiont bacteria Wolbachia in Callosobruchus maculatus populations collected from different regions (Faisalabad, Lahore, Multan, Peshawar, and Hyderabad) of Pakistan and its phylogenetic narrations. Geographical locations of the collection sites can be accessed from https://www.esri.com/en-us/arcgis. The research experiments were conducted in the Department of Zoology, Government College University, Faisalabad, Punjab, Pakistan. The samples were collected from grain storage facilities and preserved in 96% ethyl alcohol at 4°C before the molecular experiments. Rearing was also conducted under controlled laboratory conditions at 25 ± 2°C and 70 ± 5% RH. Molecular experiments were steered by using mitochondrial and three Wolbachia including two MLST genes to investigate the population dynamics of C. maculatus and Wolbachia infection status in five different localities in Pakistan. The cowpea weevil (C. maculatus) was identified using molecular DNA barcoding techniques and morphological characteristics under a microscope.
2.1. DNA extraction
Twenty two to twenty four individuals from each locality were utilized for DNA extractions through extraction kits (Sangon Biotech, China) by following the protocols provided by the product company. DNA quantification was performed by the nanodrop method. The DNA of the samples was precipitated with ethanol for purification purposes following the standardized protocol.
2.2. Polymerase Chain Reaction (PCR)
Samples were amplified through the 12S rRNA gene with primer sets (S2 Table). The samples of C. maculatus populations were passed through PCR tests for barcoding and phylogenetic analysis with the cytochrome oxidase I gene in a mixture of 25 μL with both forward and reverse sets of primers (S2 Table). The genes characterization was performed in 25 μL volume reactions containing 2.5 μL of 10× PCR buffer (Fermentas), 2.0 μL MgCl2 (2.5 mM), 0.2 μL dNTPs (200 μM), 1 μL of Taq Polymerase (1U/μL), 1 μL of each primer, 1 μL of extracted DNA, and 16.3 μL of dsH2O. PCR was conducted for 2 min at 95°C to characterize the COI mitochondria. Thereafter, under a temperature profile of 95°C for 10 min followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min and a final extension lasting 15 min at 68°C. The 12S rRNA gene fragment amplification was carried out at initial denaturation at 94°C for 15 min, followed by 45 cycles of denaturation at 94°C for 45 s, annealing at 42°C for 45 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min. During the amplification of the coxA (MLST) gene, the PCR process started at 95°C for 2 min, then it went through 32 cycles of 30 s at 94°C, 45 s at 55°C, 1 min at 72°C, and a final extension lasting 15 min at 68°C. The temperatures (Tm) for the ftsZ (53°C) and wsp (55°C) genes were maintained (S2 Table). In horizontal gel documentation, DNA fragments were used with a 1X TAE running buffer for the bands’ image pattern. Gels with 1–2% agarose and 3–4 μL ethidium bromide were utilized. DNA bands were observed on a UV transilluminator. PCR conditions, primer details, Tm information, obtained products of all tested five genes (COI, 12S rRNA, wsp, coxA, and ftsZ) are mentioned in the supplementary (S2 Table). Due to products of variable size and low titer Wolbachia density the PCR products of wsp gene from one representative sample of each population were cloned following the procedures of Arthofer et al. [47]. Plasmid DNA was purified using a Qiagen kit, and Sanger sequencing was performed using the ABI 3730 DNA analyzer (Applied Biosciences, Foster City, CA, USA). The obtained plasmids were manually edited, aligned using ClustalW [48] and compared with Wolbachia sequences from GenBank using BLAST analysis.
2.3. Phylogenetic sequence data analysis
The retrieved sequences from the present study were compared with different other sequences from Genbank data of C. maculatus within the same gene and from different countries. Wolbachia sequences from (wsp, coxA, and ftsZ) genes were compared with GenBank data of the same genes from different origins and Wolbachia strains to identify Wolbachia supergroup and strain diversity. Nucleotide sequences from the gene bank NCBI database were aligned and compared for phylogenetic tree analysis through ClustalW version 2.0.9 [48–50]. The tree topologies were analyzed by using the Neighbor-joining (NJ) implementation with the Tamura-Nei genetic distance model of Geneious software 2024 R11 version (https://www.geneious.com) and MEGA2 version 4.0 [51, 52]. Bootstrap values were assembled on 500 replicates. Genetic distances and pairwise % identity/similarity matrix (percentage of bases/ residues which are identical) were calculated using Geneious software [51]. Additionally, during the comparative analysis, we also calculated the minimum genetic distance and the maximum (%) identity of the sequences. The Bayesian inference (BI) method was performed using the MrBayes v. 3.2.6 plugin in Geneious 6.1.8 [51], following the procedures outlined by Ren et al. [72].
2.4. Sequencing and nucleotide sequence accession numbers
Three genes wsp, coxA and ftsZ) were amplified and sequenced from the populations containing Wolbachia. Cytochrome c oxidase subunit I (COI) and 12S rRNA genes were also sequenced for molecular identification and barcoding of C. maculatus. PCR products were run through an ABI 3730 DNA analyzer in both directions. After manual processing, the original sequences were assembled using the GP 2024 v R11 software. Nine sequences of five genes were deposited to the Genbank database under accession numbers (12S rRNA; PP209111, OQ934051, PP212965), (COI: PP188367, PP177610, PP188097), (coxA: PP331220), (ftsZ: PQ375108) and (wsp: PQ375109).
2.5. Statistical analysis
Statistical significance of the differences in populations between different groups was evaluated using Chi-squared tests in R 4.4.1 [53]. The null hypothesis (H0) presumed that the variables were independent, and the significance level was accustomed at 0.01. Percentage of the amplifications was calculated by using the formula (Amplified individuals (n)/total tested (N2) × 100. Significance of infection frequencies was assessed using univariate analysis of variance. This was followed by a post hoc Tukey’s HSD multiple comparison analysis at a significance level of 0.05 conducted by using the R software [53].
3. Results
3.1. Molecular identification and phylogenetics of the host Callosobruchus maculatus
The PCR with the 12S rRNA and cytochrome c oxidase subunit I (COI) genes using 116 samples from various locations resulted in an overall amplification of 94.85% and 78.48% for C. maculatus populations, respectively (S1 Table).
The amplification rates varied from 70.83% to 100% for both tested genes. The highest amplification rates for the 12S rRNA gene were observed in the populations of Faisalabad and Peshawar, followed by Lahore, Multan, and Hyderabad. On the other hand, the highest amplification rates for the cytochrome oxidase subunit I (COI) gene were observed in Lahore, followed by Multan, Peshawar, Hyderabad, and Faisalabad (F1,9 = 18.59, p < 0.0026). The sequences of 12S rRNA (279–360 bp) and COI (554–570 bp) were obtained. The retrieved sequences from the present study based on both genes were compared with already published sequences of C. maculatus from GenBank and witin the same gene.
According to the analysis of the 12S rRNA gene sequences, it was found that the sequences retrieved closely resemble the 12S rRNA sequences of C. maculatus from the GenBank data. These sequences were compared to sixteen other C. maculatus sequences from different origions within the same gene from the GenBank, and neighbor-joining (NJ) trees phylogeny were constructed. The genetic distances and % identity matrix showed that the studied populations (PP209111, PP212965, OQ934051) of C. maculatus were clustered together with the C. maculatus sequences in the GenBank data. The population (PP209111) is closely related with the populations (India, KY856743, GD 0.02, identity 99.7%). Whereas (PP212965) was closely clustered with (France, AF004131, GD 0.02, identity 99.33%) Further thethe population (OQ934051) was closely clustered with the population ((India, KY856743, GD 0.02, identity 99.64%) presented in (Fig 1; S3 Table).
Bootstrap digits were constructed on 500 replicates (> 50%). Accession numbers are mentioned with the sequence.
The sequence analysis based on COI gene exhibited that studied sequences were exactly resembled the COI sequences of C. maculatus from the GenBank data. These sequences were compared to the twenty one C. maculatus and other Coleoptera sequences of different countries within the same gene from the GenBank. Neighbor-joining (NJ) trees phylogeny were constructed. The investigated statistics (genetic distances, % identity matrix) exhibited that the studied populations (PP177610, PP188367, PP188097) of C. maculatus were exactlt clustedred with the C. maculatus population from other countries from GenBank data. These populations were closely related with the population (India, MK496677, GD 0.01, identity 99.12–100%) presented in (Fig 2; S4 Table).
Bootstrap digits were constructed on 500 replicates (> 50%). Accession numbers are mentioned with the sequence.
3.2. Screening of Wolbachia in Callosobruchus maculatus
The screening of 116 samples resulted 38.41% (wsp), 33.18% (coxA), 31.59% (ftsZ) were found positive for Wolbachia infection (S1 Table). The infection rates ranged from 18.18% to 54.16% across all three tested Wolbachia genes. The highest infection rates were observed in populations of Faisalabad, followed by Lahore, Hyderabad, Multan, and Peshawar (Chi-squared test: 3.968, p-value < 0.0464).
A phylogenetic analysis was conducted to compare the identified Wolbachia strain in C. maculatus with 30 other strains, including those from different origins documented in GenBank. The genetic identity of the Wolbachia in C. maculatus was determined by aligning and analyzing the wsp gene sequences with Wolbachia sequences from 14 A, 15 B, and 01 F supergroups from various origins. The analysis of the wsp gene sequences revealed the presence of one distinct Wolbachia strain clustered in supergroup B (Fig 3).The sequence (PQ375109) revealed the lowest genetic distance within supergroup B with accession numbers AB545609 (GD 0.04; identity 93.63%) originated from Japan. (Figs 3 and 4; S5 Table).
Map created with (ArcGIS; Esri, Tom Tom, Garmin, FAO, NOAA, USGS). Locations: Faisalabad (FSD), Multan (MLN), Peshawar (PHR), Hyderabad (HYD) and Lahore.
Bootstrap digits were constructed based on 500 replicates (> 50%) shown adjacent to the branches of clades. Accession numbers and Wolbachia supergroups are mentioned in the sequences. KR706527 Cimex lectularius (wCim) used as outgroup.
The prevalence of Wolbachia in C. maculatus populations and comparison with other Wolbachia strains from different Wolbachia harboring populations of various origins were accomplished based on two MLST gene (coxA and ftsZ) data.
Samples of C. maculatus from various populations were examined for Wolbachia infection by analyzing the coxA gene. A phylogenetic analysis was conducted to compare the identified Wolbachia strains with 22 other strains from different origins documented in GenBank. The analysis showed that the strain identified as PP331220 belongs to supergroup B with moderate to high bootstrap values (53–100%). Comparison of the Wolbachia strains exhibited that studied strain named (wCmac1) from C. maculatus was closely related with (DQ832301, GD 0.03, identity 97.51%, Tribolium confusum) and (FJ390243, GD 0.03, similarity 97.51%, T. confusum) presented in (Fig 5; S6 Table).
Bootstrap digits were constructed based on 500 replicates (> 50%) shown adjacent to the branches of clades. Accession numbers and Wolbachia supergroups are mentioned in the sequences. FJ390247 Folsomia candida used as outgroup.
The samples of C. maculatus were tested for Wolbachia infection using the ftsZ one of the five houskeeping (MLST) genes. The comparision analysis with 21 other Wolbachia sequences within the same gene from different origins documented in GenBank revealed that the strain (PQ375108) belongs to supergroup B (Fig 6) with moderate to high bootstrap values (52–100%). Furthermore, analysis exhibited that studied strain from C. maculatus was closely related with (KC305361, GD 0.02, identity 99.39%, Tribolium confusum) presented in (Fig 6; S7 Table). Based on the analysis of three Wolbachia genes the identified strain named wCmac1 according to Wolbachia nomenclature as it was found to be distinct from other C. maculatus strains.
Bootstrap digits were constructed based on 500 replicates (> 50%) shown adjacent to the branches of clades. Accession numbers and Wolbachia supergroups are mentioned in the sequences. AY764283 Zootermopsis angusticollis used as outgroup.
4. Discussion
Molecular DNA based identification using mitochondrial genes is an effective technique for identifying insect species and distinguishing between closely related species [32, 54, 55]. It is more reliable than conventional morphological identification, which can be time consuming and technically challenging. Cytochrome oxidase subunit I (COI) based DNA barcoding has been useful in identifying many cryptic and sibling species [56, 57]. It is therefore considered that molecular identification techniques are more reliable than conventional methods of morphological identification [58].
Research has shown that DNA-based barcoding using mitochondrial genes is a quick and accurate method for identifying arthropod specimens [32, 34, 58]. Both approaches can be utilized depending on the available resources and the environment. In the present study molecular identification of C. maculatus was successfully demonstrated using both COI and 12S rRNA genes.
Wolbachia is endosymbiotic bacteria that is widely present in various insects, nematodes, mites, springtails, spiders, crustaceans, bivalves, and tardigrades [24, 59–65]. It has a close relationship with mitochondrial genotypes within species and is typically transmitted vertically from one generation to the next. However, horizontal transfer may also play a role in its spread within a species. For example, Wolbachia was found to spread horizontally when infected and uninfected Trichogramma wasp larvae shared the same host [66]. Additionally, hymenopteran parasitoids of Drosophila species acquired Wolbachia through frequent horizontal transmission [67].
Wolbachia, a bacterium primarily transmitted maternally in arthropods [29], can manipulate the host’s reproductive system through cytoplasmic incompatibility (CI) and colonize new mitochondrial lineages [24, 60, 68]. Maternal transmission occurs primarily through the cytoplasm of eggs [29]. There are recognized monophyletic lineage groups labeled A to R [43, 69] with supergroup G present in Australian spiders [70].
The presence of Wolbachia in insects has been identified using different marker genes, but the reliability of these markers varies depending on the insect species [60, 71]. The wsp gene evolves more quickly among various Wolbachia strains, making PCR amplification less reliable when using current primer pairs [39, 72]. Although the wsp method is commonly used for diagnosing A and B group Wolbachia [49, 73] its potential for strain characterization is limited due to frequent recombination [74]. Nevertheless, wsp remains one of the most polymorphic markers available for detecting Wolbachia diversity and is widely used as the starting point for large screening experiments. To address the frequent recombination issue of wsp gene, two genes of multilocus sequence typing (MLST) based on five housekeeping genes has applied to explore Wolbachia diversity. MLST is a reliable tool for studying population genetics and molecular evolution, helping to minimize errors caused by frequent recombination observed in wsp [39]. Positive amplification of Wolbachia in C. maculatus populations was found based on wsp, along with two genes (coxA and ftsZ) of MLST, providing preliminary evidence of Wolbachia infection in C. maculatus. The present identified strain belonging to supergroup B, and this strain is also distinct from the already published Wolbachia strains in Callosobruchus species.
Kajtoch and Kotásková [23] thoroughly reviewed Wolbachia in Coleopteran hosts, briefing on a single Wolbachia strain in 43 species, two strains in 10 species, and multiple infections in 9 species. Previous research has also looked into the Wolbachia strains in Callosobruchus species. For example, Kondo et al. [75] examined the wsp and ftsZ genes to investigate Wolbachia in C. chinensis, C. analis, and C. latealbus. In another study, Kageyama et al. [76] used wsp genes to analyze the Wolbachia strains in C. analis and C. chinensis. In Japanese populations of C. chinensis, the Wolbachia strains wBruCon and wBruOri were found to have almost 100% infection frequencies and to cause cytoplasmic incompatibility [77]. Furthermore, Kondo et al. 76] also reported the 100% infection frequencies of wCana1 in C. analis and wClat2 in C. latealbus. Two distinct Wolbachia strains have been identified in C. analis, (non-CI-inducing wCana1 and CI-inducing wCana2. Field-collected C. analis individuals were either singly infected with wCana1 or double infected with wCana1 and wCana2. [75, 78, 79].
No previous literature was found on Wolbachia infection in C. maculatus. Therefore, by comparing the available data of Callosobruchus species it is stated that current findings on Wolbachia strains in C. maculatus populations are new and have not been characterized before using a multiple gene amplification approach.
Mitochondrial DNA and Wolbachia have the same cytoplasmic pathway of transmission. The present research revealed the phylogenetic interpretation of cowpea weevil with Wolbachia amplification and the effects of the host mitochondrial DNA evolution and diversification [80]. In the current investigation, low titer Wolbachia density were found with the all three Wolbachia genes. Previous studies have found that Wolbachia can be hard to detect due to low bacterial densities [71]. It seems that all species in a diverse group of insects are infected with Wolbachia, but the infections are sometimes at low density or only affect some individuals within a population. Bordenstein et al. [81] suggested that low Wolbachia densities might be caused by high densities of its associated bacteriophage.
Molecular phylogenetic analysis revealed that C. maculatus harbors a Wolbachia bacterium from supergroup B, which is one of the identified supergroups of arthropods [24]. This distinct Wolbachia lineage in C. maculatus populations was verified by analyzing three Wolbachia gene sequences with strong bootstrap values. Additionally, various types of endosymbiotic microorganisms have also been found in related Coleopteran families, such as Chrysomelidae, Curculionidae, and Rhynchophoridae [23].
This is the first report about the presence of Wolbachia in C. maculatus populations. Previous research suggested that Wolbachia was also identified in three species of the genus Callosobruchus [75, 76]. These findings suggest that it is likely that Wolbachia was developed by a common ancestor of C. maculatus, probably from either a phylogenetically distinct arthropod or an unevaluated bruchid carrier. The evolutionary history of Wolbachia may have occurred through interspecific transmissions [82].
It is widely believed that Wolbachia is transmitted vertically to the next generation of hosts. This is because it is commonly found in reproductive tissues. However, recent research has shown that Wolbachia can be present in various tissues throughout the host [83], which is consistent with our findings that we were able to detect Wolbachia in the genomic DNA of the entire insect, not just the reproductive tissues. Previous studies have reported the rate of Wolbachia infection in different insect populations [84, 85], which is influenced by factors such as the cost of infection to the host, the effectiveness of vertical transmission, the initial frequency of infection, and the degree of cytoplasmic incompatibility [84, 86].
To better understand the factors responsible for complete septicity in C. maculatus, it is important to explore certain elements in depth. In order to achieve this, we amplified two mitochondrial genes for host C. maculatus and three Wolbachia genes for the endosymbiotic bacteria. The study of C. maculatus provides efficient approaches to comprehend the interface and dynamics between Wolbachia and its host insect. However, in the future, we will analyze the other Wolbachia genes to study the phylogenetic alterations of Wolbachia in C. maculatus from different geographical populations.
Wolbachia manipulates its host’s biology through various means, including (CI) cytoplasmic incompatibility [24]. Melanic mutations cause a decline in host fitness [78], and intraspecies variation in cytoplasmic incompatibility intensity also plays a role in host manipulation [79]. Neverthless, the incompatible insect technique (IIT) uses Wolbachia to induce conditional sterility in mass-reared infected males crossed with wild females, reducing the target population over time. Studies have shown that IIT effectively controls insect disease vectors and agricultural pests [25, 26, 28].
This is the preliminary study which provides new insights into Wolbachia infection in C. maculatus populations, which has been rarely studied. Factors affecting the symbiotic relationship between arthropods and their symbionts include the cost of infection for hosts, spread of symbionts between insects, and environmental factors like temperature and natural enemies [87, 88]. Excessive use of pesticides, pollution, and changes in environmental factors significantly impact the decrease in mitochondrial DNA [89–91]. The low prevalence of Wolbachia in Callosobruchus species may be due to high temperatures in tropical and temperate regions [92]. Future studies should consider these factors as they may significantly affect Wolbachia diversity and focus.
5. Conclusions
The results of the present study suggest that Wolbachia infection has been found in the populations of C. maculatus. The mitochondrial phylogenetic analysis of the host species C. maculatus populations correctly revealed the molecular identification and clustered with the GenBank data from various origins of the same host and the same gene. The screening of Wolbachia in C. maculatus was accomplished through multiple gene analysis, resulting in the identification of one new strain named wCmac1, belonging to supergroup B. This study will help us understand the dynamics of Wolbachia in C. maculatus and develop effective approaches for pest management.
Supporting information
S1 Table. Samples collections, locations and screening of the Callosobruchus maculatus.
https://doi.org/10.1371/journal.pone.0313449.s001
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S2 Table. Primers used, PCR details and obtained products of 12S rRNA, mitochondrial (CO1), wsp and (MLST) genes coxA and ftsZ.
https://doi.org/10.1371/journal.pone.0313449.s002
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S3 Table. Pairwise genetic distance and percent identity values based on the 12S rRNA gene sequences with GenBank data.
https://doi.org/10.1371/journal.pone.0313449.s003
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S4 Table. Intra and inter specific analysis of pairwise genetic distance and percent identity values based on COI gene sequences with GenBank data.
https://doi.org/10.1371/journal.pone.0313449.s004
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S5 Table. Intra and inter specific analysis of pairwise genetic distance and percent identity values based on wsp gene sequences with GenBank data.
https://doi.org/10.1371/journal.pone.0313449.s005
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S6 Table. Intra and inter specific analysis of pairwise genetic distance and percent identity values based on coxA gene (MLST) and comparison with GenBank data.
https://doi.org/10.1371/journal.pone.0313449.s006
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S7 Table. Intra and inter specific analysis of pairwise genetic distance and percent identity values based on ftsZ gene (MLST) and comparison with GenBank entries.
https://doi.org/10.1371/journal.pone.0313449.s007
(XLSX)
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