Introduction
The sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) is a polyphagous pest of several host Poaceae species that commonly affects sugarcane plantations throughout the American continent, mainly in Brazil, the world's largest sugarcane producer (Rossato et al., Reference Rossato, Costa, Madaleno, Mutton, Higley and Fernandes2013; Ferreira et al., Reference Ferreira, Santana, Santos, Santos, Lôbo and Fernandes2018). Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) is the most widely used biological control agent for population regulation of D. saccharalis. In Brazil, C. flavipes is used on approximately 3.5 million hectares for the control of sugarcane borer, representing one of the most efficient applied biological control programmes in the world (Parra and Coelho, Reference Parra and Coelho2019; Fontes et al., Reference Fontes, Pires and Sujii2020). This successful control of D. saccharalis using C. flavipes shows the importance of measures to maintain the quality of this biological control agent and its host during the production process. Several factors can affect the fitness of insects; association with symbiotic organisms, for instance, has great ecological and evolutionary consequences for host species (Harris et al., Reference Harris, Brennan, Keddie and Braig2010; Dicke et al., Reference Dicke, Cusumano and Poelman2020).
Symbiosis is a broad term used to define the association between two or more species, and the effect of the symbiont on the host may be beneficial (mutualism), neutral (commensalism), or harmful (parasitism) (de Bary, Reference de Bary1879). Insects are natural hosts of numerous symbiotic microorganisms, and this association may have obligatory ecological and biological functions essential for survival – or facultative – often infecting only part of the population, being maintained through the provision of conditional benefits, or by manipulating the host's reproduction. The elimination of facultative symbionts often results in little or no apparent cost or benefit to the host insect (Douglas, Reference Douglas1989; Duron et al., Reference Duron, Bouchon, Boutin, Bellamy, Zhou, Engelstädter and Hurst2008; Brownlie and Johnson, Reference Brownlie and Johnson2009).
Symbiotic interactions between polydnaviruses and Cotesia spp. are commonly reported, with the former acting as important immune suppressors, allowing the development of immature parasitoids within the host (Stoltz and Vinson, Reference Stoltz and Vinson1979; Herniou et al., Reference Herniou, Huguet, Thézé, Bézier, Periquet and Drezen2013; Cônsoli and Kitajima, Reference Cônsoli and Kitajima2017; Tan et al., Reference Tan, Peiffer, Hoover, Rosa, Acevedo and Felton2018). Infection by the intracellular parasite Nosema sp. (Microspora: Nosematidae) has also been reported in D. saccharalis artificial rearings, and it affects the use of C. flavipes as a biological control by altering its biological parameters and search behaviour (Simões et al., Reference Simões, Reis, Bento, Solter and Delalibera2012). This shows the relevance of understanding symbiotic interactions and the effect of these microorganisms on the biological and morphological aspects of the association between C. flavipes populations. Thus, the objective of the present study was to identify facultative symbionts of the genera Wolbachia, Arsenophonus, Cardinium, Rickettsia, and Nosema in C. flavipes and the host D. saccharalis, as well as to evaluate the influence of their infection on the fitness of these hosts.
Materials and methods
Breeding and bioassays were conducted under controlled conditions at a temperature of 25 ± 1°C, relative humidity of 60 ± 10%, and a 12 h photophase in the laboratories of the Department of Crop Protection, São Paulo State University, School of Agronomic Sciences, Botucatu, São Paulo, Brazil.
Obtaining and multiplying insects
The D. saccharalis and C. flavipes individuals used in the assays were obtained from the São Paulo biofactory, Brazil. Diatraea saccharalis were fed an adapted version of the artificial diet proposed by Hensley and Hammond (Reference Hensley and Hammond1968) (agar was replaced with carrageenan), and breeding methodologies for both species followed those of Garcia et al. (Reference Garcia, Botelho and Macedo2009).
Detection of symbionts
Genomic DNA extraction
Larvae of the host D. saccharalis and adults of C. flavipes (n 50 for each species) were randomly selected from the breeding material stored in the AGRIMIP laboratory (FCA/UNESP) for genomic DNA extraction and standardisation via polymerase chain reaction (PCR). The D. saccharalis larvae were washed with saline solution (0.85% NaCl) followed by 70% alcohol and subsequently macerated in a sterile 10 ml glass beaker, after which parts of the insect's body were removed. The remaining body content was dissolved in 80 μl of Chelex100 resin (Bio-Rad Laboratories, California, US) at 10%, diluted in sterile water, and dissolved in 8 μl of proteinase K (20 μg ml−1) in 200 μl microtubes. For DNA extraction from C. flavipes, a similar procedure was performed, except that all adults of C. flavipes individuals were directly macerated in 200 μl microtubes, without removing parts of the insect's body. All tubes containing the larvae of D. saccharalis and the adults of C. flavipes were then vortexed for 5 s in a Vortex Biomixer MOD QL901, centrifuged at 6200 rpm in a MiniStar mini centrifuge, and transferred to an Infinigen thermocycler (TC-96CG) at 95°C for 20 min.
Polymerase chain reaction (PCR)
PCR and mass sequencing amplification targeting the small subunit region of ribosomal RNA were performed using specific primers for symbionts of the genera Wolbachia, Arsenophonus, Cardinium, Rickettsia, and Nosema (table 1). For the detection of all the genera except Nosema, the PCR mixture contained 12.5 μl of Taq DNA Polymerase (NeoBio), 7.5 μl of milli-Q water, 1.0 μl of each primer, and 3.0 μl of DNA sample, for a total volume of 25 μl. For Nosema, the PCR mixture contained 12.5 μl of Taq DNA Polymerase (NeoBio), 5 μl of milli-Q water, 1.25 μl of each primer, and 5.0 μl of DNA sample, for a total volume of 25 μl.
Primers used for detecting symbionts of Diatraea saccharalis (Lepidoptera: Crambidae) and Cotesia flavipes (Hymenoptera: Braconidae).
The PCRs were performed in an Infinigen thermocycler (model TC-96CG), under the following conditions: Arsenophonus, initial denaturation at 95°C for 2 min, followed by 30 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 5 min (Thao and Baumann, Reference Thao and Baumann2004); Cardinium, initial denaturation at 95°C for 2 min, followed by 30 cycles at 92°C for 30 s, 57°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min (Zchori-Fein and Perlman, Reference Zchori-Fein and Perlman2004); Rickettsia, initial denaturation at 95°C for 2 min, followed by 30 cycles at 92°C for 30 s, 58°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min (Gottlieb et al., Reference Gottlieb, Ghanim, Chiel, Gerling and Portnoy2006); Wolbachia, initial denaturation at 95°C for 3 min, followed by 30 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and final extension at 72°C for 5 min (Heddi et al., Reference Heddi, Grenier, Khatchadourian, Charles and Nardon1999); Nosema, initial denaturation at 95°C for 4 min, followed by 45 cycles at 95°C for 1 min, 48°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 4 min (Vossbrinck et al., Reference Vossbrinck, Baker, Didier, Debrunner-Vossbrinck and Shadduck1993).
PCR products were read in a UV light transilluminator (Major Science) using a 100-bp molecular marker (Norgen) and a 1% agarose gel containing 80 ml of TBE buffer solution, 0.8 g of agarose (Neo3Bio), and 0.4 μl of GelRed DNA intercalant (NeoBio).
DNA purification and Sanger sequencing
PCR products in which symbionts were detected were purified using a Cellco purification kit, according to the manufacturer's recommendations. Quantitative analyses were performed by optical density and spectrophotometry (NanoDrop MD-1000 UV-Vis). The amplified fragments were sequenced using an automatic Sanger sequencer (Model: ABI 3500, Applied Biosystems) at the Biotechnology Institute (Instituto de Biotecnologia, IBTEC) of UNESP, Botucatu, São Paulo, Brazil. The obtained sequences were compared and deposited in the GenBank database (National Center for Biotechnology Information, NCBI) using the Basic Local Alignment Search Tool (BLAST), and specific identification was performed based on sequence similarity scores and percentage of similarity.
Post-symbiont lineages production of Cotesia flavipes
After confirming infection by Wolbachia in C. flavipes, individuals were selected for the production of sister lineages without symbionts. Some of the individuals were fed pure honey to maintain the association with Wolbachia (W+), while the other group was fed honey supplemented with antibiotics (0.25% tetracycline) to eliminate the symbiont (W−) (Li et al., Reference Li, Floate, Fields and Pang2014). The parasitoids (W−) were maintained in a biological oxygen demand incubator at 28 ± 1°C under a relative humidity of 70 ± 10% and a 12 h photoperiod. The process of eliminating Wolbachia was conducted for four consecutive generations, after which individuals from both populations were randomly selected to confirm the absence of the symbiont using PCR, following the aforementioned methodologies. After this decontamination process, the W− population was fed for ten generations with pure honey to allow a complete restoration of the intestinal microbiota and eliminate all side effects of the treatment.
Wolbachia infection in C. flavipes populations (W+ and W−) was investigated before and after the experiments to validate the results obtained, as the populations of the parasitoid and the host D. saccharalis were evaluated for infection by Nosema sp., as this microorganism is commonly reported in parasitoids that multiply in D. saccharalis, causing deleterious effects in the infected population (Simões et al., Reference Simões, Reis, Bento, Solter and Delalibera2012; Paes et al., Reference Paes, Carvalho, Souza, Wilcken and Bueno2019)
Changes in the fitness of Cotesia flavipes associated with Wolbachia infection
Diatraea saccharalis larvae suitable for parasitism (4th instar) were selected from the breeding material stored at AGRIMIP (FCA/UNESP) and placed with adult W+ and W− C. flavipes females (48 h old) to allow the occurrence of parasitism. Then, the larvae were individually placed in plastic capsules (3 × 7 cm, diameter × height) with a refeeding diet (Hensley and Hammond, Reference Hensley and Hammond1968). Sixty larvae were replicated for each population of the parasitoid for evaluations of biological characteristics, flight capacity, and morphometry.
Biological aspects
The characteristics of C. flavipes were evaluated after the parasitoid larvae exited the body of D. saccharalis and during pupation. Twenty pupal masses from each population (W+ and W−) were then removed, placed in glass tubes (2.5 × 1 cm, diameter × height), and observed daily to determine the egg–pupa and pupa–adult development period (days), pupal viability (equation 1), female proportion (equation 2), and adult survival.
The survival of adult males and females was evaluated by randomly selecting one individual of each sex from each replicate, totalling 20 individuals of both sexes, placing them in individual glass tubes (2.5 × 8 cm, diameter × height), and observing them daily until death. Adults were fed thin lines of pure honey and inserted into these tubes using an entomological pin.
Flight capacity
The flight capacity of W+ and W− C. flavipes was evaluated following the methodology of Dutton and Bigler (Reference Dutton and Bigler1995) and adapted by Prezotti et al. (Reference Prezotti, Parra, Vencovsky, Dias, Cruz and Chagas2002). The test units for flight capacity consisted of a PVC cylinder internally covered with black cardboard and with the bottom sealed with black paper adjusted on a 1 cm-thick Styrofoam disk with the same diameter as the cylinder. In the test unit, an entomological glue ring was painted over an acetate strip (1 cm thick) 3.5 cm from the lower end of the cylinder, acting as a barrier for walking parasitoids. The upper part of the test unit was sealed with a transparent Petri dish internally covered with entomological glue, which acted as a trap for the parasitoids in flight.
A glass tube (2.5 × 8 cm, diameter × height) containing a pupa mass with approximately 100 ready-to-emerge C. flavipes pupae was fixed at the centre of the bottom of the test unit, on the Styrofoam disk. Inside the tube, droplets of pure honey were provided as food for the parasitoids. A total of 20 test units were used for each parasitoid population (W+ and W−), which were maintained in a vertical laminar flow chamber under fluorescent light for 3 days. After this period, the number of C. flavipes specimens in the glue ring (walkers), the Petri dish (flyers), and the bottom of the cylinder (non-flyers) was determined.
Morphometry
Ten W+ and W− adult females and ten W+ and W− males were measured. The parasitoids were placed on slides containing alcohol gel, positioned in a right-side view, and photographed using a Leica EZ4 D optical microscope coupled to a camera. The following structures were measured in ImageJ 2.00: (1) total length (thorax + abdomen); (2) length of the right forewing; (3) width of the right anterior wing; and (4) length of the posterior tibia (from the junction of the tibia with the tarsus).
Statistical analysis
The data resulting from the tests were assessed through exploratory analyses for an evaluation of the assumptions of normality and homogeneity of variances using the Shapiro‒Wilk (P < 0.05) and Bartlett (P < 0.05) tests, respectively. The egg–pupa and adult–pupa development periods, viability, female proportion, flight test results, and morphometric data were analysed by t tests (P < 0.05). The differences in flight capacity among the categories (flying, walking, and non-flying insects) were evaluated by analysis of variance and compared by Tukey tests (P < 0.05). Kaplan–Meier survival curves were generated from survival data and compared using the logRank test (P < 0.05). All analyses were performed using Minitab software.
Results
Diatraea saccharalis and Cotesia flavipes symbionts
In the present study, no associations between the studied symbiont species and D. saccharalis were found. However, the α-proteobacterium Wolbachia was detected in C. flavipes (GenBank accession number: OR074180), with a sequencing coverage of 98% identity for Wolbachia (closest GenBank accession: CP037426.1)
Changes in the fitness of Cotesia flavipes associated with Wolbachia infection
Biological aspects
No significant differences were detected in the egg–pupal development period (days) (t = 1.897, df = 1, P = 0.061; table 2), adult–pupal development period (t = 0.193, df = 1, P = 0.847; table 2), pupal viability (t = −0.658, df = 1, P = 0.512; table 2), or female proportion (t = 0.468, df = 1, P = 0.641; table 2) between the two populations.
Development (days) of egg–pupa and adult–pupa, viability (%), and female proportion of Cotesia flavipes (Hymenoptera: Braconidae) infected (W+) and not infected (W−) with Wolbachia
Survival analysis revealed no difference in the longevity of males between the two populations, with a mean survival of 48 h (χ2 = 0.190, df = 1, P = 0.663; fig. 1b). However, significant differences were observed in the survival of females in the two populations (χ2 = 11.598, df = 1, P < 0.001; fig. 1a). Females without Wolbachia had a mean survival of 72 h, which was longer than that of females with the symbiont (48 h) (fig. 1a).
Survival of Cotesia flavipes (Hymenoptera: Braconidae) infected (W+) and not infected (W−) with Wolbachia. Females (W+ and W−) (A) and males (W+ and W−) (B). The experimental conditions were: 25 ± 1°C, relative humidity of 60 ± 10%, and a 12 h photophase (LogRank test, P < 0.05).
Flight capacity
No significant differences were observed in the percentages of the flyer (t = −0.89, df = 1, P = 0.3776; fig. 2), walker (t = 0.75, df = 1, P = 0.4587; fig. 2), and non-flyer (t = −0.85, df = 1, P = 0.4749; fig. 2) adults between the W+ and W− populations (fig. 2). However, the number of flyers was greater than that of walkers and non-flyers in all populations (W+: F = 138.22, df = 2, P ≤ 0.0001; W−: F = 233.00, df = 2, P ≤ 0.0001; fig. 2).
Percentages of flyer, walker, and non-flyer adults of Cotesia flavipes (Hymenoptera: Braconidae) previously infected (W+) and not infected (W−) by Wolbachia. Box plots represent the median and median quartiles of 75 and 25%, whiskers represent upper and lower bounds, and dots represent value discrepancies. The experimental conditions were: 25 ± 1°C, relative humidity of 60 ± 10%, and a 12 h photophase. Columns with the same lowercase letter (among populations) and capital letter (within population) are not significantly different from each other (t test, P < 0.05 and Tukey test, P < 0.05, respectively).
Morphometry
No significant differences were detected in body length (t = −1.169, df = 1, P = 0.258; fig. 3) or right tibia length (t = −1.482, df = 1, P = 0.156; fig. 3) between the female C. flavipes W+ and W− populations. However, compared with W+ females, C. flavipes W− females had longer right-wing lengths (t = −2.449, df = 1, P = 0.025) and wider right-wing lengths (t = −2.742, df = 1, P = 0.013) (fig. 3).
Body length, right-wing length, right-wing width, and right tibia length (all in mm) of females of Cotesia flavipes (Hymenoptera: Braconidae) infected (W+) (black boxes) and not infected (W−) (white boxes) with Wolbachia. Box plots represent the median and median quartiles of 75 and 25%, respectively; whiskers represent upper and lower bounds, and dots represent outliers. The experimental conditions were: 25 ± 1°C, a relative humidity of 60 ± 10%, and a 12 h photophase. The different letters within each graph indicate significant differences (t test, P < 0.05).
Uninfected males of C. flavipes (W−) had a longer right tibia length than individuals infected with Wolbachia (W+) (t = −2.70, df = 1, P = 0.015; fig. 4). No significant differences were detected in the other morphological measures between males of the two populations (body length: t = 0.285, df = 1, P = 0.779; right-wing length: t = −1.820, df = 1, P = 0.085; right-wing width: t = −1.799, df = 1, P = 0.089; fig. 4).
Body length, right-wing length, right-wing width, and right tibia length (all in mm) of males of Cotesia flavipes (Hymenoptera: Braconidae) infected (W+) (black boxes) and not infected (W−) (white boxes) with Wolbachia. Box plots represent the median and median quartiles of 75 and 25%, respectively; whiskers represent upper and lower bounds, and dots represent outliers. The experimental conditions were: 25 ± 1°C, a relative humidity of 60 ± 10%, and a 12 h photophase. The different letters within each graph indicate significant differences (t test, P < 0.05).
Discussion
Symbiotic associations between Wolbachia and Cotesia spp. have already been reported in the literature, but the associations between Wolbachia and C. flavipes are presented herein for the first time (Mochiah et al., Reference Mochiah, Ngi-Song, Overholt and Stouthamer2002; Branca et al., Reference Branca, Le Ru, Vavre, Silvain and Dupas2011, Reference Branca, Le Ru, Calatayud, Obonyo, Musyoka, Capdevielle-Dulac, Kaiser-Arnauld, Silvain, Gauthier, Paillusson, Gayral, Herniou and Dupas2019; Rattan et al., Reference Rattan, Hadapad, Reineke, Gupta and Zebitz2011; Srinivasa et al., Reference Srinivasa, Rajeshwari, Venkatesan and Baby2011; Murthy et al., Reference Murthy, Venkatesan, Jalali and Ramya2015). Wolbachia is a genus of Gram-negative, non-spore-forming, obligate intracellular symbiont bacteria that infects a wide range of arthropods. Wolbachia can be transmitted vertically from host females to offspring by being loaded into the egg (O'Neill et al., Reference O'Neill, Hoffmann and Werren1997; Duron et al., Reference Duron, Bouchon, Boutin, Bellamy, Zhou, Engelstädter and Hurst2008). Some studies have also reported the horizontal transfer of Wolbachia across populations (O'Neill et al., Reference O'Neill, Giordano, Colbert, Karr and Robertson1992; Werren et al., Reference Werren, Zhang and Guo1995). Nevertheless, as no infections of this symbiont were observed in D. saccharalis, Wolbachia transmission in C. flavipes was herein demonstrated to occur vertically.
Wolbachia infection mainly affects the host's ecological aspects and reproductive system, inducing cytoplasmic incompatibility, parthenogenesis, feminisation, and annihilation of males, which results in reproductive isolation of the infected population and, consequently, successful establishment of the symbionts in host population (Stouthamer et al., Reference Stouthamer, Breeuwer and Hurst1999; Mochiah et al., Reference Mochiah, Ngi-Song, Overholt and Stouthamer2002). Although some Wolbachia strains successfully infect host populations, others cannot parasitise the reproductive systems of their host or adequately do so (Hoffmann et al., Reference Hoffmann, Clancy and Duncan1996). This may explain the findings of the present study, as feminisation and annihilation of males infected by Wolbachia were not observed herein, suggesting that this symbiont cannot induce thelytokous parthenogenesis in this population and/or cannot replicate at high levels in the body of this parasitoid. Although Wolbachia-induced parthenogenesis in C. flavipes was not observed herein, future investigations should evaluate the effect of bacterial infection on the intraspecific cytoplasmic incompatibility of this insect; a mixture of infected and uninfected populations with such reproductive changes would have a decreased population growth rate, which would have implications for biological control programmes.
The present study also detected possible deleterious effects of the symbiotic association on the biological and morphological aspects of C. flavipes. Uninfected females had longer right-wing lengths and wider right-wing lengths and were longer lived, and uninfected males had longer tibia lengths than males infected with Wolbachia. The results presented here are supported by several reports of deleterious effects on the fitness of arthropods infected with Wolbachia (Fry et al., Reference Fry, Palmer and Rand2004; McGraw and O'Neill, Reference Mcgraw and O'Neill2004; Serga et al., Reference Serga, Maistrenko, Rozhok, Mousseau and Kozeretska2014; Stevanovic et al., Reference Stevanovic, Arnold and Johnson2015; Zhou et al., Reference Zhou, Shang, Liu, Zhang, Huo, Zhang and Dong2023).
Considering that the longevity and morphology of parasitoids are considered indicators of the quality of these control agents (Sagarra et al., Reference Sagarra, Vincent and Stewart2001; Wang and Keller, Reference Wang and Keller2020), the information from this study is the basis for understanding the impact of costs of Wolbachia infection in C. flavipes; therefore, measures are needed to investigate infections by this symbiont in large-scale production of C. flavipes to maintain the production of efficient individuals for use in augmentative biological control programmes.
Such effects on the fitness of arthropods can be reversed by treating the infected population with antibiotics, as observed in C. flavipes. Dedeine et al. (Reference Dedeine, Vavre, Fleury, Loppin, Hochberg and Boulétreau2001, Reference Dedeine, Bouletreau and Vavre2005) reported the participation of this bacterium in the oogenesis of Asobara tabida Nees (Hymenoptera: Braconidae), with post-symbiont females being unable to produce mature oocytes and, therefore, reproducing; this was the first record of a transition from facultative to obligatory symbiosis in arthropod associations. Nevertheless, no such effect of Wolbachia infection was found herein, as the post-symbiont C. flavipes populations showed no reproductive changes.
Conclusions
The results of the present study can be used as a basis for understanding the role of Wolbachia in the quality of C. flavipes populations, in addition to demonstrating the importance of studies on these microorganisms that influence their host's biological, physiological, and reproductive characteristics.
Acknowledgments
The authors gratefully acknowledge the financial support for this research by the following organisation and agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES; grant code 001).
Competing interests
None.
