The group of cryptic species of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) comprises more than 40 whitefly species (Elfekih et al., 2021; Campbell et al., 2023). In Uganda, 13 B. tabaci species have been identified infesting cassava, Sub-Saharan Africa (SSA), from 1 to 13 (SSA1-13). Five of these species (SSA9 to SSA13) were recently discovered based on mitochondrial cytochrome c oxidase Subunit 1 (mtCO1) analysis (Mugerwa et al., 2021a; Mugerwa et al., 2021b). Additionally, other B. tabaci species, including East Africa 1 (EA1), Indian Ocean (IO), and MED, have also been reported on cassava, albeit in low numbers (Mugerwa et al., 2021a). These different species within the B. tabaci complex can exhibit variations in their biology, behavior, host preferences, and ability to transmit plant viruses (Omondi et al., 2005; Civolani et al., 2014; Ning et al., 2015). Based on mtCO1 analysis, SSA1 was further subdivided into sub-groups, ranging from SSA1-SG1 to -SG5, also referred to as mitotypes. The SG1 and SG2 mitotypes were recently shown to belong to the same B. tabaci SSA1 species and renamed SSA1 SG1USG2 (Campbell et al., 2023).

Bemisia tabaci SSA1 SG1USG2 is an important pest affecting cassava and other crops in Uganda and other countries, causing damage by feeding on plant sap and reducing growth, yield, and crop quality (Kalyebi et al., 2018). Moreover, it is a vector for plant viruses causing economically important cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) epidemics in Uganda (Maruthi et al., 2002; Colvin et al., 2006; Maruthi et al., 2017). In this work, we will refer to them as SSA1, SSA1-SG1, and SSA1-SG2 to denote the two mitotypes belonging to SSA1.

Outbreaks, defined as the presence of more than 100 whiteflies on the top five leaves of cassava plants, have persisted in Uganda since the 1990s (Legg et al., 2002; Legg et al., 2014; Macfadyen et al., 2018), impacting both small-scale and large-scale cassava production. Various factors have been suggested as drivers of increasing whitefly infestations, including monoculture cropping (Macfadyen et al., 2018), as well as the mutually beneficial interaction between cassava mosaic viruses and whiteflies or the enhanced genetic fitness of certain whiteflies on cassava (Legg et al., 2002).

Bemisia tabaci species carry primary and secondary endosymbionts, and the presence and dynamics of these endosymbionts can vary based on factors like spatiotemporal changes (El Hamss et al., 2022), life stage (El Hamss et al., 2024), and/or the host plant (Maruyama et al., 2020).

Bemisia tabaci species harbor endosymbiotic bacteria, which reside in the body cavity, haemolymph, or intracellularly in specialized cells called bacteriocytes (Ghosh et al., 2018). Endosymbionts can enhance the fitness of B. tabaci by improving food supply through the synthesis of essential nutrients, such as carotenoids (Sloan and Moran, 2012). Endosymbionts may also protect against natural enemies by increasing resistance to parasitoids or predators (Giorgini et al., 2023). Portiera aleyrodidarum, is a primary endosymbiont present in all B. tabaci species, including the SSA ones, and which supplements their amino acid-deficient diets. Secondary endosymbionts, such as Rickettsia, Wolbachia, and Arsenophonus, are present in SSA1 either in single or mixed infections (Ghosh et al., 2015). While Hamiltonella, Cardinium, and Fritschea have not yet been reported in SSA1 so far. Some secondary endosymbionts were shown to influence fitness and reproduction in SSA species, and in particular within SSA1 (Ghosh et al., 2018; El Hamss et al., 2021). We analyzed the microbiome of SSA1 whiteflies at two levels: (i) endosymbionts, previously defined as intracellular bacteria, and (ii) the meta-microbiome, consisting of all other bacteria, including those unreported or not previously known in Hemipteran insects. Due to their distinct characteristics, these groups were analyzed separately. Unlike endosymbionts, meta-microbiome bacteria can also be transmitted horizontally. This approach ensures a clear distinction, allowing for a more precise analysis of their respective patterns of variation. To investigate microbiome differences associated with whitefly outbreaks, we compared the microbiome profiles of adult B. tabaci SSA1 collected from outbreaking and non-outbreaking cassava regions in Uganda. Deep-sequencing techniques provided insights into microbiome variations and their potential relationship with whitefly outbreaks.

The central research question was whether the outbreaking status of cassava whiteflies is associated with differences in their microbiome. To minimize environmental variability, all samples were collected from cassava fields located within a 5 km radius near Lake Victoria, ensuring relatively similar agro-ecological conditions across sites. Sampling focused on a single host plant (cassava), and sites were categorized based on whitefly abundance: outbreaking sites had more than 100 whiteflies per top five leaves, while non-outbreaking sites had substantially fewer individuals. This design allowed us to explore potential microbiome associations with outbreak status under comparable environmental settings.

In this study, we investigated the microbiome composition and diversity between outbreaking and non-outbreaking SSA1 B. tabaci populations on cassava in Uganda. Significant variations were demonstrated between endosymbionts and meta-microbiome composition, diversity, and structure between the outbreaking and non-outbreaking sites of SSA1-SG1 and SSA1-SG2 mitotypes.

Endosymbiont diversity was not influenced by outbreaking status, whereas the meta-microbiome was structured by outbreaking status. A specific 20 core ASVs were unique to the outbreaking SSA1-SG1, including different strains of Caldalkalibacillus, and species belonging to Bacillaceae, and Micrococcaceae. Although SSA1-SG1 and SG2 are the same species (Ally et al., 2019; Mugerwa et al., 2021b; Ally et al., 2023; Campbell et al., 2023), we found that their microbiome networks, diversity, and composition differed significantly. It is not uncommon to observe variations within populations, where one population may be more ‘aggressive’ or ‘fit’ than the other, as seen in studies with fruit flies or other insects such as the harlequin ladybird Harmonia axyridis (Vandereycken et al., 2012). These variations within populations can trigger invasive behavior, and they are often linked to ecological and evolutionary factors that influence fitness and adaptability. This highlights the importance of considering population-level differences when studying invasion dynamics, moving beyond taxonomic classifications to understand the biological drivers of outbreaks.

We examined two levels of microbial diversity: endosymbionts and meta-microbiome. Endosymbionts, primarily Portiera, Arsenophonus, and Wolbachia, showed no variation by site or outbreak status, aligning with mitochondrial profiles. Candidatus hemipteriphilus was found in 1.7% of samples, and Rickettsia in 3.5%, with 9 haplotypes identified. These findings are consistent with previous studies demonsarting that endosymbiont associations tend to be conserved, not only at the mitotype level but also across different whitefly species beyond Uganda (Kanakala and Ghanim, 2019). A large-scale study of over 2000 whiteflies revealed a non-random distribution of facultative endosymbionts among distinct B. tabaci species (Zchori-Fein et al., 2014). Arsenophonus was primarily found in the Mediterranean (MED) species complex and Middle East-Asia Minor 2 (MEAM2), whereas Wolbachia was absent in MEAM1 individuals (Zchori-Fein et al., 2014; Wang et al., 2019).

In our study, the contrasting patterns observed between endosymbionts and the environmentally acquired meta-microbiome likely reflect differences in their modes of transmission and ecological roles. The endosymbionts, which are maternally inherited, showed relatively stable distributions across both outbreaking and non-outbreaking populations, suggesting they are conserved components of the whitefly’s internal microbiota. This stability implies a foundational role in host biology, potentially linked to essential functions such as nutrient provision or reproductive manipulation.

Beyond endosymbionts, we found significant differences in the diversity, composition, and network properties of the meta-microbiome between SSA1-SG1 and SG2, indicating that microbiome variability exists even within the same species. Genetic makeup and outbreak status likely influence this variation. Given that these bacteria are environmentally acquired, their composition is likely shaped by local factors such as cassava phyllosphere communities, climatic conditions, or insect density. This variability may reflect a more flexible, short-term form of microbial association, potentially enabling whiteflies to respond rapidly to environmental stressors or host plant defenses during outbreaks. The clearer structuring of the meta-microbiome by outbreak status, compared to endosymbionts, supports the idea that environmentally acquired bacteria might contribute to outbreak dynamics by influencing host adaptability or competitive success under high-density conditions.

To better understand the role of these different strains, deeper sequencing with longer fragments is needed.

This study highlights distinct meta-microbiome structures in SSA1 whiteflies from outbreak sites, emphasizing the role of specific bacterial groups in whitefly performance. Twenty ASV groups, including Bacillus, Caldalkalibacillus, and Micrococcaceae, were predominantly found in SSA1-SG1 individuals from outbreak sites, suggesting their potential involvement in local adaptation and/or increased fitness. Caldalkalibacillus (ASV3 and ASV4) and Micrococcaceae (ASV1 and ASV2) were also abundant and cosmopolitan across all tested categories, suggesting that these taxa might not be directly influenced by outbreak status or linked to a specific mitotype. Their roles in their host biology should be further investigated. The gut microbiome are known to play a key role in helping whiteflies to adapt to different plants, potentially enhancing their performance in sites with favorable environmental conditions (Su et al., 2016; Santos-Garcia et al., 2020). For instance, an increase in Mycobacterium bacteria in whiteflies feeding on pepper helped them to break down harmful substances, improving their survival (Santos-Garcia et al., 2020). Similarly, in aphids transferred to different Solanaceae plants, a decrease in Buchnera symbionts and an increase in Pseudomonas were observed, suggesting that microbiome shifts may be linked to host plant adaptability. However, aphids on pepper showed a smaller increase in Pseudomonas, potentially affecting their ability to thrive on this plant (He et al., 2021). It is worth noting that in our study, all whiteflies were collected from different sites but from the same host plant, cassava, within a monoculture agricultural system in Uganda. This reduces the likelihood that the observed microbiome variations were due to host plant adaptation. Instead, the differences may reflectsite-specific environmental factors or intrinsic microbiome community dynamics. Might be influencing the outbreak patterns. In microbial ecology, the complexity of co-occurrence networks is often linked to ecological stability and resilience. In our study, the microbial network in SSA1-SG2 was notably more complex than in SSA1-SG1. This greater connectivity may indicate a more robust microbial structure, potentially buffering the host population against perturbations such as pathogen invasion or environmental stress. Such resilience could contribute to the lower outbreak tendency observed in SSA1-SG2 populations. Further studies are needed to confirm whether this network complexity supports greater niche stability or functional redundancy in the whitefly microbiome.

A related study by Li et al. (2023) on wheat microbiome found that surface-associated (phyllosphere) communities were more complex and stable than internal (endosphere) ones, with key bacterial taxa like Pantoea, Massilia, and Pseudomonas structuring the community. While direct comparisons between plant and insect microbiomes are limited by biological differences, this highlights a broader ecological principle: microbiome complexity can reflect and influence host-environment interactions. Given that whiteflies are sap-feeders, the microbiomes of both the insect and its host plant may interact. Future investigations into the cassava microbiome may reveal how plant-associated microbes influence whitefly microbial communities, potentially shaping outbreak dynamics and ecological adaptation.

This study examined the link between outbreaking whiteflies and their meta-microbiome, uncovering non-random differences in bacterial communities between outbreaking and non-outbreaking sites. Co-occurrence analysis identified ecological factors shaping the microbiome in SSA1. B. tabaci likely acquires Caldalkalibacillus from environmental sources, which may assist in digestion or stress resistance (de Jong et al., 2024). The presence of 10 Caldalkalibacillus strains in outbreak sites suggests a potential role in outbreak dynamics.

While bacteria like Bacillaceae and Pseudomonadaceae are traditionally seen as pathogens for their hosts (Deka et al., 2022), our findings suggest they may have a positive effect in outbreaking populations. However, this effect could also be the opposite—we do not know for certain. It is possible that in sites with high whitefly densities, these bacteria act as pathogens, spreading rapidly within the population due to the close proximity of thousands of individuals during outbreaks. This study provides a snapshot of the whitefly microbiome during a defined outbreak event in Uganda, using a geographically and environmentally constrained sampling strategy to reduce confounding variables. Although sampling was limited to a single country and time point, the observed patterns of microbial diversity and composition. We acknowledge that broader temporal and spatial sampling, as well as functional validation experiments, would help establish causality and generalize findings across regions. Nevertheless, our results offer important insights and generate hypotheses for future research on the ecological role of microbiomes in whitefly outbreaks.

This also raises the question of whether these bacterial taxa could also contribute to the eventual decline of an outbreak. Given that we collected samples at a single time point and from a limited number of insects, we must be cautious in extrapolating our results to the broader population. Additionally, considering that these bacterial genera are known pathogens, multiple scenarios should be explored to fully understand their role in whitefly outbreaks.

Previous studies on multicolored Asian lady beetle (Harmonia axyridis) demonstrated that Micrococcaceae may enhance nutrient absorption during oviposition, potentially aiding in the digestion of cell walls and lignocelluloses (Hu et al., 2018; Huang et al., 2022). The Micrococcaceae family is frequently found in the midguts of a wide range of insect species (Uzmi et al., 2019; Hathnagoda et al., 2024). For example, the genus Micrococcus, Micrococcus luteus was detected in both the pre-pupal and adult stages of the dissected gut of a subcortical beetle (Agrilus planipennis) (Vasanthakumar et al., 2008). In our study, this bacteria was detected at the family level with different strains observed, longer sequences are also needed to identify them at the species level to see which species are present in SSA1 whiteflies. Micrococcaceae can be transferred to whiteflies while feeding on sap leaves or while coming in contact with an infected environment but this should be investigated.

On a broader scale, our results support the observation that differences in outbreak dynamics shape the meta-microbiome of SSA1 whiteflies. Its diversity and composition differed significantly between non-outbreaking and outbreaking populations across all sites. Future studies on the drivers of local invasions or epidemic emergence should consider the microbiome as a potential contributing factor, with a focus on identifying the specific bacteria or bacterial combinations involved.