
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39237643
70755
10.1038/s41598-024-70755-2
Article
Impact of diet and bacterial supplementation regimes on Orius strigicollis microbiota and life history performance
Hung Yi-Ting 1
Wong Adam Chun-Nin 2
Tang Cheng-Kang 3
Wu Ming-Cheng mcwu@nchu.edu.tw

1
Tuan Shu-Jen sjtuan@dragon.nchu.edu.tw

1
1 grid.260542.7 0000 0004 0532 3749 Department of Entomology, National Chung Hsing University, Taichung, Taiwan, Republic of China
2 https://ror.org/02y3ad647 grid.15276.37 0000 0004 1936 8091 Department of Entomology and Nematology, University of Florida, Gainesville, FL USA
3 https://ror.org/05vn3ca78 grid.260542.7 0000 0004 0532 3749 Program in Plant Health Care, Academy of Circular Economy, National Chung Hsing University, Nantou, Taiwan, Republic of China
5 9 2024
5 9 2024
2024
14 2072714 6 2024
20 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Given the growing interest in manipulating microbiota to enhance the fitness of mass-reared insects for biological control, this study investigated the impact of an artificial diet on the microbiota composition and performance of Orius strigicollis. We compared the microbiota of O. strigicollis fed on an artificial diet and moth eggs via culturing and 16S rRNA gene amplicon sequencing. Subsequently, we assessed life history traits and immune gene expression of O. strigicollis fed on the artificial diet supplemented with Pantoea dispersa OS1. Results showed that microbial diversity remained largely unaffected by the artificial diet, with similar microbiota compositions in both diet groups. OS1, a minor member of the microbiota but significantly enriched in bugs fed on the artificial diet, improved nymphal survival rates and shifted adult longevity-reproduction life history in females. Additionally, OS1 supplementation elevated the transcription of antimicrobial peptide diptericin. According to population parameters, the group receiving OS1 only during the nymphal stage showed higher population growth potential compared to the group supplemented across all life stages. These findings reveal the resilience of O. strigicollis microbiota under distinct dietary conditions and highlight the potential of using natural symbionts and specific supplementation regimes to improve Orius rearing for future biocontrol programs.

Keywords

Antimicrobial peptide
Diptericin
Pantoea dispersa
Artificial diet
Probiotic
Subject terms

Entomology
Computational biology and bioinformatics
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Orius strigicollis (Poppius) (Hemiptera: Anthocoridae) is a commercialized biological control agent with great predation efficiency against pest arthropods, including thrips, aphids, mites, and whiteflies on various cropping systems1,2. Both their natural prey and eggs of Cadra cautella (Walker) (Lepidoptera: Phycitidae) are commonly used as feed sources during rearing, but the labor-intensive and time-consuming nature of utilizing these feed sources has limited market potential. To enhance effectiveness, mass-rearing on artificial diet has been proposed3–5. Our previous research demonstrated that O. strigicollis can be successfully sustainable reared on a meridic artificial diet (without insect components), with cost-effectiveness comparable to its natural prey, Tetranychus urticae Koch (Acarina: Tetranychidae)3. Moreover, this artificial diet has been demonstrated to support successive generations of O. strigicollis, thereby providing evidence of its viability for a sustainable rearing program (Supplementary Table S1).

Aligning with the ongoing efforts to optimizing mass rearing of Orius, targeted manipulation of the gut microbiota has been proposed as a potential strategy6,7. The gut microbiota plays a pivotal role in enhancing various functions and overall fitness of insect hosts4,8,9. Additionally, diet is a key factor affecting the composition and structure of the gut microbiota10,11, and diet-microbiota interactions can impact multiple aspects of the host's life history, including survival, development, and reproduction12. A healthy gut microbiota can also aid in defense against pathogens and boost immunity, while dysbiosis of the gut microbiota has been associated with reduced health and diseases13–15. Therefore, targeted microbiota manipulation in mass rearing holds the potential to restore beneficial gut microbial communities, thereby mitigating fitness decline, enhancing pathogen resistance, and ultimately bolstering pest control effectiveness. For example, Somerville et al.16 showed improved larval growth and male performance in the diamondback moth (Plutella xylostella), a commonly mass-reared insect used in sterile insect technique for pest management, by supplementation with Enterobacter cloacae isolated from moth’s gut. Other bacteria have also demonstrated similar beneficial effects, with Enterobacter sp. enhancing development in Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), Enterobacter sp. AA26 increasing pupal and adult production in Bactrocera oleae (Rossi) (Diptera: Tephritidae) and Asaia sp. boosting development in Aedes aegypti larva (L.) (Diptera: Culicidae)8,17–19. While the efficacy of utilizing bacteria as probiotics has been demonstrated in other insects, the effects of artificial diet on O. strigicollis microbiota and the potential benefits of microbial supplementation to the host remain unclear.

To gain deeper insights into the symbiotic bacteria of Orius species raised in artificial settings, we studied the microbiota of O. strigicollis fed both the artificial diet and eggs of C. cautella. This investigation led to the isolation and identification of bacteria from female O. strigicollis, in with we focused on an isolate (a Pantoea strain) for subsequent bacterial supplementation experiment, as Pantoea was previously reported to have beneficial associations with other insects20,21. We evaluated the effects of diet and bacterial supplementation on O. strigicollis life history traits, including survival rate, fecundity, and population parameters. Additionally, we assessed the immune response by measuring the mRNA expression levels of antimicrobial peptides (AMPs), which are commonly used as markers to elucidate immunomodulatory effects. Our findings underscore the potential of harnessing symbionts and targeted supplementation to enhance Orius mass rearing for biocontrol programs.

Results

Composition and diversity of microbiota in O. strigicollis reared on moth eggs and artificial diet

The microbiota of O. strigicollis adults reared on moth eggs (ME) and the artificial diet (AD) were identified using 16S rRNA gene amplicon sequencing. High-throughput sequencing generated a total of 282,801 effective reads, with each sample containing between 51,268 and 44,929 reads. These reads were subsequently clustered into 118,997 operational taxonomic units (OTUs, clusters of sequencing reads that differ by less than 3% in similarity in this study). Based on phylogenetic classification using 16S rRNA gene database, the majority of reads were identified as belonging to the phylum Proteobacteria (74.5–99.8% and 83.0–96.4% of OTUs in O. strigicollis reared on moth eggs and the artificial diet, respectively; Fig. 1a). Within Proteobacteria, the two most dominant classes in O. strigicollis were Gammaproteobacteria and Alphaproteobacteria (Fig. 1b). While Gammaproteobacteria remained highly abundant across both diets (60.9–84.5% and 81.3–93.8% in ME and the AD group), the artificial diet treatments reduced the abundance of Alphaproteobacteria (13.5–19.3% to 1.7–3.7%). At the family level, Enterobacteriaceae was dominant under both diets, accounting for 58.8–84.5% and 57.5–83.9% in the ME and AD groups, respectively. The genus Izhakiella, which was most abundant in the family Enterobacteriaceae, dominated both groups, except for the AD3 treatment (81.0–90.8% and 78.5–88.0% in the ME and AD groups, respectively; Fig. 1c). AD3 showed a distinct dominated genus that also belonged to the family Enterobacteriaceae, identified as Serratia spp. (62.1% in total OTUs of AD3; Supplementary Table S3). Within Alphaproteobacteria in the ME group, Gluconacetobacter dominated in ME1 and ME2 (0.9–14.3%), while Wolbachia dominated in ME3 (0.9–16.9%).Fig. 1 Relative abundances of bacteria in Orius strigicollis fed on moth eggs and artificial diet (a) at Phylum level, (b) at Class level, (c) at Genus level. AD1-AD3: Females fed on the artificial diet, with three independent biological replicates. ME1-ME3: Females fed on moth eggs, with three independent biological replicates.

The second most abundant phylum in O. strigicollis was Firmicutes (0.1–14.8% and 1.6–16.1% in O. strigicollis reared on ME and AD, respectively), most of which were in the class Bacilli (1.3–14.4% and 0.9–16.0%, respectively), family Staphylococcaceae (0.1–12.6% and 0.1–15.8%, respectively), and the genera Staphylococcus was more abundant in ME1 and AD3 (12.6% and 15.9%). The core microbiota of O. strigicollis from both dietary groups, comprising the top 20 abundant taxa, showed a 90% similarity. This core microbiota primarily consists of Izhakiella spp., Serratia spp., Staphylococcus spp., Gluconacetobacter sp., Kocuria sp., endosymbionts Wolbachia spp., and Pantoea sp. The diversity of bacterial communities is reflected through the Shannon diversity and Faith’s phylogenetic diversity indices, and community richness is reflected by the Chao1 index. While the AD group generally showed higher diversity indices and observed OTUs, no statistically significant difference was observed between the groups (Fig. 2a–d; p > 0.05 in Kruskal–Wallis test). This suggests similar OTUs richness and diversity, with potentially higher variation in the ME group.Fig. 2 Comparison of alpha diversity metrics between microbiomes in Orius strigicollis fed on moth eggs (ME, orange) and artificial diet (AD, green). (a) Box-whisker plots of the numbers of observed operational taxonomic units (OTUs), (b) Shannon diversity, (c) Faith’s phylogenetic diversity, and (d) Chao1 diversity. OTUs is defined as clusters of sequencing reads that differ by less than 3% in similarity. Points represent individual samples. Lines in the boxes correspond to the median of samples. Statistical significance was analyzed by Kruskal–Wallis test. n.s., not significant (p > 0.05).

To identify bacterial taxa with significant differences between the two diet groups, linear discriminant analysis (LDA) effect size (LEfSe) was used to pinpoint distinct taxa at various taxonomic levels (kingdom, phylum, class, order, family, genus, and species) between two groups. Our analysis revealed 9 differential taxa in the AD group and 8 in the ME group (Fig. 3a; LDA score > 2 or < − 2). The presence of only 17 taxa identified as significantly different by the LEfSe analysis suggests that the diets may have had a limited impact on the diversity of bacterial species in the Orius. However, the differential taxa observed between the ME and AD groups imply that the diets influence the abundance of bacteria from different classes. Within the ME group, two taxa were assigned to the Alphaproteobacteria category, collectively representing 16% of the total OTUs. Conversely, in the AD group, the taxa were distributed across Gammaproteobacteria, Saccharimonadia, and Campylobacteria, with abundances each lower than 5% (Fig. 3b). These findings suggest that compared to the AD, the ME had a greater capability to increase the abundance of specific bacterial taxa, particularly Wolbachia and Gluconacetobacter. Besides, albeit to a relatively minor extent, Pantoea, with a relative abundance of 0.72%, was enriched in the AD group compared to the ME group (Fig. 3b,c).Fig. 3 LEfSE analysis. (a) The bar plots represent the significantly differential taxa between Orius strigicollis fed on an artificial diet (AD; green) and moth eggs (ME: orange), based on effect size (LDA score [log 10]). Differences among classes were obtained by the Kruskal–Wallis test (p < 0.05). (b) The relative abundance of significantly differential taxa between AD group and ME group. (c) Cladogram showing different abundant taxa between AD group and ME group. Each small circle represents a taxon at a different taxonomic level, with the diameter of the circle proportional to its relative abundance. Different colors represent different groups, and nodes with different colors represent the communities that play an important role in the group represented by the color. Differences among classes were obtained by the Kruskal–Wallis test (p < 0.05).

Identification of isolates

A total of ten bacterial colonies, cultured from females fed on the ME, were chosen for sequencing and identification based on their distinct morphologies. BLAST analysis of the partial 16s rDNA sequences of these isolates were assigned to the genera lzhakiella, Paenibacillus, Pantoea, Staphylococcus, Bacillus, Mammaliicoccus, Enterobacter, and Serratia (Supplementary Table S4). Bacterial strain isolated from Orius strigollis, named as OS1 to OS11, and their details can be found in the Supplementary Table S4 (online only). The phylogenetic analysis in Fig. 4 confirms that the OS1 belongs to the genus Pantoea and is closely related to P. dispersa LMG 2603, with a 16S rDNA identity of 99.5%.Fig. 4 Phylogenetic tree of the OS1 isolate used as probiotics in this study constructed using the sequences of 16S rDNA. The OS1 is labeled as a black spot. Sequences for the 16S rDNA phylogenetic analysis were obtained from the GenBank database for Pantoea species and their closest phylogenetic neighbors. Escherichia coli was used as an outgroup organism. The GenBank accession number is included in the bracket before the bacterial scientific name. This analysis involved 22 nucleotide sequences.

Abundance of Pantoea in O. strigicollis and their immune genes expression after P. dispersa OS1 supplementation

Based on real-time qPCR of the 16S rRNA gene, the abundance of Pantoea within the female O. strigicollis receiving the OS1 supplement increased by 16.7, 59.4, and 79.7 times, on days 1, 3, and 5, respectively (Fig. 5a, p = 0.0495). However, there was no significant difference in total bacterial abundance between the supplemented and control groups (Fig. 5b). These findings suggest that supplementation with the OS1 specifically boosts Pantoea levels without affecting the overall bacterial abundance.Fig. 5 Bacterial colonization levels and antimicrobial peptides relative gene expression profile of Orius strigicollis females supplied with Pantoea dispersa OS1 and sucrose solution as control. (a) The bacterial loads of Pantoea spp. were assessed through by quantitative polymerase chain reaction (qPCR) using a Pantoea specific16S rDNA primers. (b) Total bacterial loads were assessed through by qPCR using universal bacterial 16S rDNA primers. DNA samples obtained from 1, 3, and 5-day-old females fed with 5% sucrose solution (control), and the OS1 in 5% sucrose solution (P. dispersa adding) since they emerged were prepared for qPCR. Asterisks indicate statistically significant differences (*, p < 0.05) (Kruskal–Wallis test). (c) Defensin (Def) and diptericin (Dip) relative gene expression were analyzed by qPCR using the 2-△△CT method. Asterisks indicate statistically significant differences (*, p < 0.05) (Student's t-test). β-Actin are used as the internal control.

The immunomodulatory effects of the OS1 on female O. strigicollis were evaluated by measuring the transcriptional responses of two key AMPs, defensins and diptericin. These AMPs are among the most extensively studied in insects22,23. The Dip was found to be significantly elevated in the supplemented group compared to the control group (up to approximately 15.1-fold, p = 0.037, Fig. 5c), whereas the Def did not show any significant difference. The results suggest that the OS1 primarily stimulated the immune response of O. strigicollis through diptericin among the two AMPs studied.

Effect of P. dispersa OS1 supplement on life history traits

To assess the impact of the OS1 on both nymphal and adult stages of O. strigicollis, we evaluated life history traits on different supplementation regimes: the Nym group, receiving the OS1 only during the nymphal stage; the Nym + Adu group, receiving the OS1 throughout all life stages; and a control group, which was provided only with a sucrose solution as supplementation, without the OS1. Table 1 presents the life history traits (survival, adult longevity, fecundity, oviposition, and adult pre-ovipositional period) evaluated. In terms of nymphal survival rate, both the Nym and Nym + Adu groups exhibited a significant 19.3% increase in survival rate compared to the control group (p = 0.0274 and 0.0284). For adults, we observed that OS1 significantly influenced female longevity and reproduction, but the direction of effect varied based on the supplementation regime. Compared to the non-supplement control, female longevity in the Nym group was reduced, but extended in the Nym + adu group. Additionally, the female longevity in the Nym + Adult group significantly exceeded that of the Nym group by 5.1 days (p = 0.03544). However, no significant differences were observed in male longevity among the three groups. Differing from the trends observed in longevity, the fecundity and the adult preoviposition period (APOP, the duration between the adult emergence and first reproduction of female) of females from the Nym group showed an increase compared to the non-supplemented control, whereas the Nym + Adu group exhibited reduced fecundity and APOP. Specifically, females from the Nym group laid 61.5% more eggs and exhibited 2.3 days shorter APOP compared to those in the Nym + Adu group (p = 0.00997 and p = 0.0100, respectively). The results indicate that the balance between reproduction and longevity in females was contingent upon the OS1 supplementation regimens.Table 1 Biological characteristics (mean ± SE) of Orius strigicollis in different supplementation regimes.

Statistics	na	Control	n	Nym	n	Nym + Adu	
Nymphal survival rate (%)	31	77.4 ± 7.5 b	30	96.7 ± 3.3 a	30	96.7 ± 3.3 a	
Male longevity (d)	13	43.4 ± 2.9 a	14	39.4 ± 2.6 a	20	38.3 ± 2.0 a	
Female longevity (d)	11	43.1 ± 3.8 ab	15	41.1 ± 1.7 b	9	46.2 ± 1.7 a	
Fecundity (total eggs/female)	11	29 ± 6.5 ab	15	32.3 ± 2.5 a	9	20 ± 3.9 b	
APOPb (d)	9	4.0 ± 0.3 a	15	3.8 ± 0.2 a	9	6.1 ± 0.9 b	
Oviposition dayc (d)	9	17.3 ± 2.7 a	15	14.3 ± 1.3 a	9	11.3 ± 1.9 a	
Nym: tested individuals fed on the artificial diet supplemented with the OS1 sucrose solution for only the nymphal stage; Nym + Adu: both nymphs and adults fed on the artificial diet supplemented with the OS1 sucrose solution; Control: tested individuals fed on the artificial diet supplemented with 5% sucrose solution alone at all life stages. Standard errors were estimated using 100,000 bootstrap samples. Means followed by the same letters in a row are not significantly different among the treatments, as determined by evaluating bootstrap percentile confidence intervals with a significance level of 5%. an = number of recorded individuals. bAPOP: Adult preoviposition period, defined as the duration between the adult emergence and first reproduction of female. cOviposition day: the exact period during which oviposition took place.

Table 2 presents the population parameters of O. strigicollis, evaluating the growth potential of the population from two supplemented groups and a control group. The intrinsic rate of increase and finite rate of increase in the Nym group were 1.7 and 1.0 times higher, respectively than those observed in the Nym + Adu group. Additionally, the net reproductive rate in the Nym group was 2.7 times higher than in the Nym + Adu group. These parameters collectively indicate that the Orius population from the Nym group exhibited significantly higher growth potential than that from the Nym + Adu group.Table 2 Population parameters (mean ± SE) of Orius strigicollis in different supplementation regimes.

Statistics	na	Control	n	Nym	n	Nym + Adu	
Intrinsic rate of increase (r) (d−1)	31	0.0820 ± 0.013 ab	30	0.1027 ± 0.0076 a	30	0.0610 ± 0.0135 b	
Finite rate of increase (λ) (d−1)	31	1.0855 ± 0.0137 ab	30	1.1081 ± 0.0084 a	30	1.0629 ± 0.0143 b	
Net reproductive rate (R0) (offspring/individual)	31	10.3 ± 3.4 ab	30	16.1 ± 3.2 a	30	6.0 ± 2.0 b	
Mean generation time (T) (d)	31	28.4 ± 1.3 ab	30	27.1 ± 0.6 a	30	29.4 ± 1.0 b	
Nym: tested individuals fed on the artificial diet supplemented with the OS1 sucrose solution for only the nymphal stage; Nym + Adu: both nymphs and adults fed on the artificial diet supplemented with the OS1 sucrose solution; Control: tested individuals fed on the artificial diet supplemented with 5% sucrose solution alone at all life stages. Standard errors were estimated using 100,000 bootstrap samples. Means followed by the same letters in a row are not significantly different among the treatments, as determined by evaluating bootstrap percentile confidence intervals with a significance level of 5%. an = number of recorded individuals.

The age-specific survival rate (lx) showed a simplified form of the survival history and the probability that a newly-hatched nymph will survive to age x (Fig. 6a). A higher lx of O. strigicollis was observed in two of the OS1 supplement groups (the Nym and the Nym + Adu) as compared to the control group before the 40 or 43th day. The age-stage-specific fecundity (fx7) of individuals from the Nym group displayed a distinct peak between the 22nd and 25th day. In contrast, the fx7 of females from the Nym + Adu group did not exhibit a clear peak. Within the control group, fx7 peaked between the 20th and 25th day, with intermittent 1-day peaks occurring after the 40th day, attributed to a few surviving females (Fig. 6b). The results indicate that under the Nym + Adu regime, OS1 exhibited a beneficial impact on nymphal survival rates, yet subsequently resulted in a decline in female performance.Fig. 6 The age-specific survival rate (lx) (a), and age-stage-specific fecundity (fx7) (b) of Orius strigicollis fed on an artificial diet under three the Pantoea dispersa OS1 supplementation regimes. Control: Bugs were supplemented with sucrose solution. Nym: Bugs were supplemented with OS1 during the nymphal stage. Nym + Adu: Bugs were supplemented with OS1 during all stages.

Discussion

This study is the first to reveal the bacterial microbiota of O. strigicollis and how the bacterial communities respond to an artificial diet as compared to a factitious prey (moth egg), which is commonly used in the Orius mass-rearing system. Previous studies have suggested that diet significantly influences the gut microbial community and dominant flora of omnivorous insects24,25. However, our findings indicate that the diet treatments had a limited effect on the number of OTUs, diversity, or core taxa identities in O. strigicollis. Similarly, the microbiota diversity of the seven-spot lady beetle, Coccinella septempunctata (Linnaeus) (Coleoptera: Coccinellidae), showed minimal differences between groups reared on aphids and an artificial diet26. Albeit to a relatively minor extent, these differences in microbiota composition might be due to variations in the nutritional composition between natural prey/factitious prey, and artificial diets27. The absence of significant diet-driven changes in the diversity of microbiota might suggest the microbiota of O. strigicollis is resilient to dietary shifts. This resilience may be attributed to the metabolic versatility of the microbes, allowing them to thrive under the two dietary conditions28. Besides, the adaptation of O. strigicollis to the artificial diet may have been facilitated by the stable resident microbiota community.

In the microbiota of the O. strigicollis, Izhakiella was identified as the most abundant genus across both diet groups, except for one replicate of AD3 that was dominant by Serratia. Izhakiella was first reported as an Orius symbiont and it has previously been found in Pseudatomoscelis seriatus (Reuter) (Hemiptera: Miridae) as the most abundant genus29. Prior to this, Izhakiella was first identified in insects as endobacteria within Capsodes infuscatus Brulle (Hemiptera: Miridae) by Yana Aizenberg-Gershtein et al.30. While this genus has been consistently associated with Hemipteran insects, its functional role remains largely uncertain. Further research is needed to elucidate the contribution of this genus to the physiology, behavior, and overall fitness of Hemipteran hosts. Besides the predominant bacterium Izhakiella, other minor satellite bacteria, such as Kocuria and Staphylococcus, were identified in O. strigicollis fed on the artificial diet. These bacteria might have been introduced from the yolk of hen's eggs31, a natural ingredient in the diet. The impact of these bacteria on O. strigicollis remains to be elucidated through further research.

In our study, we identified and cultured ten bacterial isolates from female O. strigicollis as part of an ongoing process of screening potential probiotic bacteria. Among these isolates, we found representatives from genera such as Pantoea, Serratia, and Staphylococcus. While previous research has highlighted the beneficial roles of these genera in various insects32,33, only Pantoea has been specifically associated with beneficial effects on Hemipteran hosts. For example, in the stinkbug of the Pentatomidae family (Hemiptera: Pentatomidae), most symbionts belong to the Pantoea genus as being the midgut crypt symbionts34,35. These Pantoea bacteria can produce essential nutrients that are either absent or deficient in the host diet and cannot be synthesized autonomously, examples include Candidatus Pantoea persica in Acrosternum arabicum Wagner (Hemiptera: Pentatomidae)20 and Pantoea spp. in Brachynema germari Kolenati (Hemiptera: Pentatomidae)20,21. Moreover, based on our LEfSe analysis result, Pantoea was significantly elevated in O. strigicollis fed on the artificial diet compared to moth eggs, yet it only made up only < 5% of the total microbiota. The Pantoea was assumed to have a potentially beneficial association with Orius fed on the artificial diet. Based on the above evidence and assumption, we chose P. dispersa OS1 for further characterization on its potential effects on O. strigicollis.

The fitness benefits associated with the presence of O. strigicollis in the life table assays provide more evidence of its properties. The results suggest that the OS1 reduced the mortality of the nymph at both Nym and Nym + Adu treatments. Additionally, they provide evidence that the bacterial supplement effects highly dependent on the supplementation regime and can vary by sex. The population parameters are based on practical data and reliable statistics, providing a comprehensive and precise evaluation of population growth potential36. At the population level, the Nym group exhibited the highest growth potential. Considering the costs involved in insect rearing, a higher population growth potential, brief APOP, and intense oviposition significantly enhance the productivity and offspring collection efficiency in the mass-rearing system. From the standpoint of enhancing the efficacy of O. strigicollis mass-rearing, these results suggest that the Nym regime showed a significant advantage compared to the Nym + Adu. The beneficial effects of the bacterial supplement on the host insect were also reported by Grau et al.37, suggesting that the protective function of the probiotic is limited to nymphs. Furthermore, the researchers indicated that this process may involve the modulation of the immune system.

To confirm the possible immune system modulation caused by the OS1, the gene expression fold of Dip and Def were quantified. Our result suggests that the immune system of Orius was primed against the OS1 by increasing the abundance of diptericin. The activation of the immune response by commensal bacteria is beneficial for the survival of insects in artificial rearing setups as it helps prevent potential pathogens introduced from their food source. While pathogen resistance was not directly tested in this study, previous research suggests that gut commensal bacteria can stimulate host immune defenses by pre-activating several AMPs, thereby enhancing resistance to entomopathogens38,39. Thus, a potential explanation for the advantage conferred by OS1 on nymph survival is the pre-activation of Orius immunity.

We observed changes in longevity and reproduction in the females influenced by the OS1 supplementation regimes. One potential explanation for the observed decrease in fecundity with OS1 supplementation is that the microbe elicits an immune response, resulting in a performance cost. Immune function is costly in terms of energy expenditure40,41. Previous studies have shown evidence of trade-offs in the resources allocation between immunity and reproduction, as both processes rely on dietary protein, and protein availability can be limiting42,43. Besides, maintaining immunity might prevent infections in individuals, thereby contributing to their extended longevity44. Although the actual underlying mechanism remains unclear, our results suggest that the supplementation regime should be carefully considered when implementing bacterial complementation to enhance the performance of O. strigicollis.

It is important to note that non-sterile diets were used throughout this study to prevent alterations in the nutritional quality and physical properties of the diet components caused by sterilization. Accordingly, this study did not ascertain whether the observed differences in the microbiota between the artificial diet and moth egg groups are attributable to potential microbial variations between the dietary treatments. Additionally, the O. strigicollis used in this study have been maintained in the laboratory for several years, potentially leading to differences in their microbiota compared to natural populations. As previous studies have reported that the bacterial diversity of laboratory-reared Triatoma infestans (Klug) (Hemiptera: Reduviidae)45 and Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)46 has been reported to be significantly reduced compared to wild populations. Future research will include surveys of field-collected O. strigicollis to investigate how domestication through laboratory rearing affects microbiome composition.

Our study offers new insights into target microbiota intervention at specific life stages to improve insect mass-rearing. Our findings suggest that supplementing the OS1 during the nymphal stages would be the most effective strategy to enhance survival. The supplementation regime can be tailored to enhance specific life history traits (longevity, reproduction, etc.) and the concept is broadly applicable to other insect rearing systems. Follow-up studies will be conducted to investigate the effect on the subsequent generations of O. strigicollis. This will clarify the optimized frequency and dose of the OS1 supplementation to provide a commercial formulation for mass-rearing systems.

Methods

Insect rearing

The O. strigicollis colony was provided by the Miaoli District Agricultural Research and Extension Station, Miaoli County, Taiwan. This stock colony was maintained on an alternative prey, eggs of C. cautella, and cultured in plastic containers (37.5 × 23.5 × 15 cm) with a ventilation hole covered with nylon gauze. Soybean (Glycine max L.) seedlings were supplied to provide moisture, shelter, and oviposition substrate for O. strigicollis. The colony was kept at 25 ± 3 °C, 60 ± 15% RH, and a photoperiod of 12:12 h (L:D). To avoid the potential negative effect of long-term inbreeding, more than 100 individuals of O. strigicollis were collected from the field annually and first reared separately for more than two generations. After insectary health inspection, the new cohort was mixed with the lab-reared colony to prevent contamination by parasitoids or entomopathogens.

In an artificial diet feeding group, newly hatched neonates were randomly selected and individually fed with a piece of disposable sponge (0.25 × 0.25 × 0.25 cm) soaked with 0.02 ml of fresh diet solution in a Petri dish (9 cm in diameter). Before soaking, the sponge was sterilized with 70% ethanol and then rinsed with autoclaved distilled water. The individual was moved to a sterilized Petri dish containing a new soybean seedling daily when the artificial diet was replaced. The artificial diet used in this study was based on the combined diet 1 described by Hung et al.3 The nymph diet contained 40% fresh egg yolk (Lulon, Changhua, Taiwan), 10% yeast extract (Difco, Becton Dickinson, Rutherford, NJ, USA), 5% beef extract (Difco, Becton Dickinson), 5% sucrose (Sigma-Aldrich, St. Louis, MO, USA), 5% honey (Honey world farm, Taichung, Taiwan), and distilled water. The adult diet consisted of 30% fresh egg yolk, 5% yeast extract, 5% beef extract, 3% sucrose, 20% honey, 10% milk powder (Red Cow, E-mart Marketing Co., Taipei, Taiwan), and distilled water. To prepare the diets, the yeast extract and water were autoclaved before adding the other ingredients, and no preservatives or antibiotics were included. The artificial diet was then stored at − 20 °C until needed.

DNA extraction and 16S rRNA gene amplicon sequencing

Before DNA extraction, twenty O. strigicollis females from each feeding group (the artificial diet and moth eggs) were subjected to surface-sterilized with 75% ethanol then rinsed with deionized water three times. Total genomic DNA extraction was performed using DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA samples were quantified using QIAquick PCR Purification Kit (Qiagen), and their integrity was verified by agarose electrophoresis. The V3–V4 region of the bacterial 16S rRNA gene was amplified using primers 314 F with specific barcode and 805R (Supplementary Table S2)47. Each sample was subjected to PCR in 30 µL of the reaction mixture, including 0.5 µL of KAPA HiFi DNA Polymerase, 0.75 µL KAPA dNTP Mix, 0.5 µL KAPA HiFi Fidelity Buffer, 0.75 µL of forward and reverse primers (10 µM), and 10 ng template DNA (the reagents and mix listed above were provided by Kapa Biosystems, Woburn, MA, USA). The PCR conditions were as follows: initial denaturation at 95 °C for 3 min followed by 5 cycles of 98 °C (20 s), 57.5 °C (20 s), and 72 °C (20 s), and a post-PCR incubation at 72 °C for 3 min. Samples containing bright main bands ~ 500 bp were retained for further experiments. The PCR products were pooled and then purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). The sequencing library was generated using the Celero DNA-Seq System (Nugen, San Carlos, CA, USA), with an index code added. Finally, the library was sequenced on the Illumina MiSeq System (Tri-I Biotech Inc., Taipei, Taiwan) and 2 × 301 bp paired-end read segments were generated.

Computational analyses of 16S rRNA sequencing data

Data obtained from independent sequencing were analyzed separately using QIAGEN CLC Genomics Workbench (v 10.1.1, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Samples were marked as follows: Moth egg group (ME): O. strigicollis fed with eggs of C. cautella; Artificial diet group (AD): O. strigicollis fed with the artificial diet. Paired-end sequences imported into CLC Genomics Workbench were quality-controlled and combined. If the similarity between sequences was more than 97%, they were assigned to the same operational taxonomic units (OTUs). For the taxonomic classification of OTUs, SILVA (v132), comprehensive database of 16S rRNA sequences for microbial classification, were utilized as 16S rRNA gene databases48. Alpha diversity (the number of observed OTUs, Shannon diversity, Faith’s phylogenetic diversity, and Chao1 diversity), were analyzed using R package “microeco”49. The alpha diversity (within samples) of OTUs was analyzed through the number of observed OTUs and Chao1, Shannon, and Faith’s phylogenetic diversity indices to obtain the species richness and uniformity information in the samples, as well as to identify common and unique OTUs among different samples. Linear discriminant analysis effect size (LEfSe) was used to screen the taxa for significant differences between two groups with LDA scores greater than two. A cladogram was drawn to show the distribution of these taxa at different taxonomic levels by R package “microeco”49.

Isolation and identification of bacteria

Bacteria were identified from the 5-day to 7-day old female O. strigicollis fed on moth eggs following the below isolation and culture procedure, all conducted were carried out in biosafety cabinet using an aseptic technique. Five female O. strigicollis were surface sterilized with 75% ethanol, and placed to a sterile tube, following by rinsing three times in distilled water prior to homogenization with a plastic pestle in 200 µL double distilled water. Ten µL of the homogenate was spotted on NB/LB plates, and the plates were incubated at 28 °C for 24 h (Supplementary Table S4). Single colonies were chosen and inoculated into the liquid culture mentioned in Supplementary Table S4 to establish pure cultures, and the culture tubes were incubated at 28 °C for 16 h. The pH of all liquid media and plates was adjusted to pH 6.5 before inoculation. To preserve the bacteria, liquid cultures were cryopreserved by adding a 20% (V/V) glycerol solution and storing them at − 80 °C.

To identify isolated bacteria, a 1 mL overnight culture was transferred into a sterile 1.75 mL tube and DNA was extracted using QIAamp DNA Microbiome Kit (Qiagen) according to the manufacturer’s instructions. The 16 s rRNA was amplified with universal primers 27F and 1492R Supplementary Table S2)50. Each PCR sample had a total volume of 50 μL, contained 25 μL 2 × PCR supermix (TOOLS, Taiwan), 10 μM of primer pair, and 10 ng template DNA. The PCR amplification reaction was conducted according to described by Wu et al.51. The amplified 16S rDNA gene fragments were then cloned into a pGEM-T easy vector (Promega, USA) and transformed into competent DH5α cells according to the manufacturer’s protocol. Blue/white selection was carried out and the plasmids were isolated for bidirectional sequencing. The obtained sequences were subjected to BLAST analysis using the GenBank database established by the US National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) to allow taxonomic identification by similarity.

The 16S rDNA phylogenetic analysis of the OS1 isolate was performed for 22 nucleotide sequences, which included the sequence of the OS1 obtained in this study, as well as 10 sequences belonging to the genus Pantoea, 5 sequences belonging to the genus Kluyvera, and 5 sequences belonging to the genus Erwinia. These sequences were obtained from BLAST results in the GenBank database to perform a comparison with our sequences. In addition, the sequence of Escherichia coli (X80725.1), which was used as an outgroup, was included. Multiple sequence alignment was performed using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) program, and phylogenetic trees were constructed using MEGA11 software52. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Bootstrapping was performed for 1000 replicates.

In vivo assays

Abundance of Pantoea in O. strigicollis after supplementation

To assess the abundance of Pantoea in O. strigicollis after continually supplementation of the OS1, newly emerged females fed from moth eggs were individually placed in Petri dishes individually and fed the artificial diet supplemented with the OS1 in sucrose solution (108 bacteria per mL, Sigma-Aldrich). Prior to supplementation, the OS1 was cultured in LB media at 28 °C for 16 h, pelleted (by centrifugation for 1 min at 8000 rpm), and resuspended in 5% sucrose solution. Females fed on the artificial diet supplemented only with sucrose solution, devoid of OS1, were collected to form the control group. On day 1, 3, and 5, ten insects from each group were sampled to assess bacterial abundance using quantitative PCR (qPCR). Before DNA extraction, each female was surface sterilized with 75% ethanol and washed in sterilized deionized water three times. Total genomic DNA extraction was performed using a DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. Two primer pairs, 27F/ 355R, and Pantoea_F/Pantoea_R, were used to determine the abundance of total bacteria and Pantoea spp. in O. strigicollis (Supplementary Table S2). The abundance of Pantoea spp. was determined using the same thermal cycle conditions as described in Huang et al.53. but the annealing step was modified (60 °C for 30 s) for the total bacterial abundance assay. Using standard curves from the amplification of the cloned target sequence in a pGEM-T easy vector (Promega), we calculated the absolute DNA copy number for the reaction template and then adjusted it based on the dilution to calculate the total DNA copy number for each sample. Each qPCR experiment was performed using three independent biological replicates with three technical replicates. Biological replicates address natural variation among organisms, while technical replicates address variability in the experimental process.

Life table parameters evaluation

Newly hatched neonates were randomly collected within 12 h of emergence. Three treatment groups were designed: (1) Nym group: neonates fed on the artificial diet supplemented with the OS1 sucrose solution at their nymphal stage, while adults were fed only the artificial diet; (2) Nym + Adu group: neonates fed on the artificial diet supplemented with the OS1 sucrose solution at all life stages; (3) control group: neonates fed on the artificial diet supplemented with 5% sucrose solution alone at all life stages. The survival was recorded daily until all individuals of the tested cohorts developed into adults. Once the adults emerged, one male and one female were transferred to a new Petri dish. Stems of soybean seedlings wrapped individually with wet cotton were provided for oviposition, and egg numbers were counted daily under a dissection microscope. The female fecundity was recorded until all individuals died.

Gene expression measurement using reverse transcription-quantitative PCR (RT-qPCR)

All adults were 7 days old at the time of sampling. Five females were preserved in the RNAprotect Tissue Reagent (Qiagen) at − 20 °C freezer before homogenization for total RNA extraction. Insects were ground using a motorized hand pestle and total RNA was isolated using RNeasy Kit (Qiagen) according to the manufacturer’s instructions. Residual DNA was removed using the RNeasy columns (Qiagen) with silica-membrane technology. The pure RNA samples were then quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at − 80 °C.

The iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used to reverse transcribe 1 μg of total RNA into cDNA. Next, qPCR was performed to determine the expression of two antibacterial peptides, genes defensin (Def) and diptericin (Dip). Primers for the application of Def, Dip, and β-actin genes were designed using the gene sequences of Rhodnius prolixus (Stål) (Hemiptera: Reduviidae) and A. lucorum from the GenBank database (AY196130.1, EU448993.1, and KU188517.1) and using Primer3 software54 (Supplementary Table S2). β-actin was used as a reference gene for internal control. Each assay included a negative control without a cDNA template. Each qPCR assay was conducted in a 96-well plate, and every 20 μL of reaction solution contained 10 μL of 2 × iQ SYBR Green Supermix (Bio-Rad, Taipei, Taiwan), 2.5 μL of 1.6 μM of each gene-specific primer, and 5 μL of diluted cDNA. After the reaction agents were added, the 96-well plate was placed inside a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) and allowed to react. The reaction conditions as described in Hsu et al.55. For each cycle, the fluorescent reaction signals were detected and collected using Bio-Rad CFX Maestro software (Bio-Rad). The relative gene expression data were analyzed using the 2−ΔΔCt method56. Each RT-qPCR assay involved three independent biological replicates with three technical repetitions.

Life table performance and statistical analyses

The raw data of each O. strigicollis were analyzed according to an age-stage, two-sex life table36,57,58 using the TWOSEX-MSChart computer program59. The age-specific survival rate (lx), age-stage fecundity (fx7, where x is age and 7 is the stage of female) and life table parameters (intrinsic rate of increase; finite rate of increase; net reproduction rate; mean generation time) were calculated according to Chi and Liu57 and Chi58. The adult preoviposition period (APOP) is defined as the time from female adult emergence to its initial oviposition. Oviposition days refers to the exact period during which oviposition took place. The intrinsic rate of increase (r) was estimated using the Euler–Lotka formula through the interactive bisection method with the age indexed from 060, which quantifies the rate at which a population grows when there are no limitations on resources. The finite rate of increase (λ) is calculated as:1 λ=er

The net reproduction rate (R0) is defined as the average number of off-spring that an individual can produce throughout its entire lifespan. The mean generation time (T) means the time that a population needs to increase to R0-times its size at the stable age-stage distribution. The formulas of these parameters are defined as below:2 ∑x=0∞e-r(x+1)lxmx=1

3 R0=∑x=0∞lxmx

4 T=lnR0r

where x is age and j is stage. The standard errors of the nymphal survival rate, adult longevity, APOP, oviposition period, fecundity, and population parameters of O. strigicollis fed on the artificial diet supplemented with the OS1, and sucrose solution were calculated using the bootstrap resampling method61,62 (m = 100,000). The statistical test used for comparing these parameters involved evaluating bootstrap percentile confidence intervals, with significance determined at the 5% level61,62. The difference in alpha diversity and bacterial abundance among groups was compared using the Kruskal–Wallis test, with values of p < 0.05 considered statistically significant. For RT-qPCR, Student’s t-test were used to determine the level of significance of differences in all gene expression in relation to the bacteria addition treatment. Differences were considered significant at p < 0.05. These statistical analyses and graphs were performed using R (version 4.3.2).

Supplementary Information

Supplementary Tables.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-70755-2.

Acknowledgements

The authors would like to thank Dr. Hsin Chi (Department of Entomology, National Chung Hsing University, Taichung, Taiwan) and Dr. Ying-Tsong Chen (Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan) for advice on the statistical assistance with the two-sex life table theory and bioinformatic analyses. This study is partially supported by the National Science and Technology Council: NSTC 110-2313-B-005 -018-MY3 and the Bureau of Animal and Plant Health Inspection and Quarantine, Ministry of Agriculture: 110AS-5.3.1-BQ-B1(3) to S.J. Tuan.

Author contributions

Y.H., S.T., and M.W. conceived and designed the experiments; Y.H. performed the experiment, analyzed the data, and wrote the paper; A.W., C.T., M.W., and S.T. edited the paper, revised the version, and approved the final manuscript.

Data availability

The datasets generated and/or analszed during the current study are available in PRJNA1120647 repository. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1120647.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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