The Camellia spiny whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae) (Figure 1), is a major pest to the Theaceae plants that originated in China (Kanmiya et al., 2011; Chen et al., 2018; Andrianto and Kasai, 2022) and is currently considered to be an invasive species over the world tea producing area (Uesugi et al., 2016; Jansen and Porcelli, 2018; Adi and Susanti, 2020; Rizzo et al., 2021). The main harm caused by the A. camelliae whitefly is the sucking of tea sap and the excretion of honeydew that covers the surfaces of tea leaves which subsequently promotes the growth of sooty mold. The combination of all these factors causes a decline in the health and vigor of the tea tree and a reduction in yield (Tian et al., 2020). In fact, citrus whitefly A. spiniferus had been denoted as A. camelliae before 2011 till the Japanese researchers proved the difference between the spiny whitefly on tea trees and the whitefly A. spiniferus on citrus and defined the new scientific name of Aleurocanthus camelliae for Camellia spiny whitefly in tea plant (Kanmiya et al., 2011; Uesugi and Sato, 2011).

Insects generally carry heritable symbiotic bacteria including primary and secondary symbionts which coevolve with host insects (Wang et al., 2020), and endosymbionts play an important role in insect growth and reproduction (Hillman et al., 2017; Akami et al., 2019; Zhang et al., 2019). Cooperation between the host insect and its microbiota is largely beneficial for all partners, but shifts in microbiota composition can also violate this mutualism (dysbiosis) and result in severe adverse effects on insects (Engl and Kaltenpoth, 2018; Grenier and Leulier, 2020). It is known that Aleyrodidae species harbor endosymbiont Bacteroides playing fundamental roles in their fitness. In addition, it has been demonstrated that endosymbionts can both protect their host against enemies (i.e., parasitoids, other bacteria, and viruses) and influence some biological traits (e.g., fitness, sex ratio, and host range), usually ameliorating the growth rate of the host under certain stress conditions (Bing et al., 2013; Su et al., 2014; Bubici et al., 2020). Limited studies related to bacterial symbionts of other close species have been examined, including A. woglumi (Pandey et al., 2013) and A. spiniferus (Bubici et al., 2020). However, bacterial symbionts of A. camelliae are poorly understood except for a report on the Wolbachia infection in Aleurocanthus cryptic species complex (Andrianto and Kasai, 2022). Therefore, this study aimed to examine the infection status and diversity of symbionts and their influence on A. camelliae.

Many 16S rRNA-based surveys of intestinal microbiota use taxonomic classification as the primary tool for comparative analysis to identify taxa that are indicative of a certain host phenotype. For example, 16S rRNA gene sequencing reveals the diversity and dynamic change of bacterial symbionts of Adelphocoris suturalis (Xue et al., 2021) and a shift in the microbiota of Diaphorina citri during the psyllid life cycle (Meng et al., 2019) and environmental-associated microbiota composition in honey bees (Jones et al., 2018). However, there was a lack of systematic research on the microorganisms hosted in A. camelliae and their function. We, therefore, by using high throughput sequencing of the bacterial 16S rRNA genes, detected the composition and diversity of bacteria in the A. camelliae to grasp the resource status of bacterial microorganisms in this whitefly.

The functions of the symbionts in whiteflies have attracted increasing research efforts in recent years. For example, the Cardinium provides protection of Bemisia tabaci against heat stress and can increase the longevity of both female and male adults and oviposition periods (Yang et al., 2021). The abundance of the Portiera in the whitefly population decreased with the increase in the nitrogen levels of the host plant (Liu et al., 2020). Hamiltonella–Cardinium coinfection induced lower fecundity, egg hatchability, and a number of female offspring, leading to a male-biased sex ratio in B. tabaci (Zhao et al., 2018). In addition, Hamiltonella elimination from the whitefly host by heat treatment and antibiotics influences the sex ratio of an insect host (Shan et al., 2019). Symbiotic bacteria of whitefly together affected host resistance to neonicotinoids (Barman et al., 2022). Reductions of primary and secondary symbionts in whiteflies following an antibiotic treatment negatively affect the host's fitness (Zhang et al., 2015).

Because most of the bacterial symbionts found in whiteflies and other sap-sucking insects are uncultivable, one way to study the functions of these symbionts is to establish host lines that differ in symbiosis treatment and then compare the performance between different host lines. For this purpose, antibiotic treatment of the hosts has frequently been employed to cure symbionts (Zhang et al., 2020; Weiland et al., 2022). This approach has also been tried with whiteflies in B. tabaci (Shan et al., 2016; Lv et al., 2018; Andreason et al., 2020). The antibiotic rifampicin was used in the experiments because all the previous articles, which present successful elimination or suppression of whitefly symbionts (Shan et al., 2016; Zhao et al., 2020), report that rifampicin was the most selective and efficient agent in their experiments between the several antibiotics tested, including tetracycline and ampicillin. In this study, we examined the effects of rifampicin treatments on the demographic development of the Camellia whitefly and its symbionts, including the primary symbiont Portiera and other secondary symbionts.

Life table analysis is the most widely used method to study life history characteristics and population dynamics, which can provide a powerful tool to describe the impact of external factors on the growth, development, reproductive capacity, and population growth of insects (Chi et al., 2020; Yang et al., 2020). However, the demographic evaluation of A. camelliae and its changes caused by symbionts have not been studied so far. This research on symbionts of A. camelliae revealed the endosymbionts and their function on host reproduction, survival, population dynamics, and adaptation to the environment. It will provide new insight into the biological function of endosymbionts and the theoretical basis for the potential biological control of A. camelliae.

Bacterial symbionts play prominent roles in their insect hosts. Many reports related to other whitefly insects, such as Bemisia tabaci (Shan et al., 2016; Su et al., 2016; Hashmi et al., 2019; Zhao et al., 2020), proved the effects of symbiotic bacteria on the host growth and development. However, research on symbionts of spiny whitefly and their function was lacking. To make clear this respect, we checked the community composition and diversity of symbiotic bacteria of A. camelliae by 16S rRNA high-throughput sequencing and investigated their biological implications by using the age–stage two-sex life table with comparing the aposymbiotic and natural groups.

Our present results proved that the symbiotic bacteria of A. camelliae were abundant. Interestingly, it can be observed from the aspect of symbiotic bacteria diversity that larva and adult endosymbiont community diversities did not show obvious changes. We inferred that endosymbionts are in a relatively stable state in A. camelliae to mutualism with host insects (Harris et al., 2010). We found that the main five symbiotic bacteria genus were Portiera, Arsenophonus, Wolbachia, Rickettsia, and Pseudomonas, which together composed approximately 94.83–96.24% in A. camelliae. Those type of symbiotic bacteria composition in A. camelliae is similar to that in Bemisia tabaci (Zchori-Fein et al., 2014; Hashmi et al., 2019) though different in relative content. However, there is an exception that no symbiotic Hamiltonella was found in A. camelliae, which was reported to exist in Bemisia tabaci (Bello et al., 2019). Therefore, we deduced that insects of the same genus have similar symbiotic bacteria.

Proteobacteria are a major group (phylum) of gram-negative bacteria and most important endosymbionts and include a wide variety of bacterial genera in different insects (Yun et al., 2014). It was also found to be dominated in A. camelliae in this present result, which was similar to the findings in other Hemipteran insects (Lim and Ab Majid, 2020; Liu et al., 2022). All five main genera, Portiera, Arsenophonus, Wolbachia, Rickettsia, and Pseudomonas, belonged to the Proteobacteria phylum. Among them, Portiera as the primary endosymbiont was the most abundant genera in a stable content level throughout the development cycle of A. camellia, indicating that Portiera plays a lasting symbiotic role in the growth, development, and survival of A. camellia. Portiera can evade autophagy of the whitefly bacteriocytes by activating the TOR (target of rapamycin) pathway by providing essential nutrients (Wang et al., 2022). Interestingly, Portiera can facilitate the whitefly performance by cooperation with the panBC gene for pantothenate production (Sun et al., 2022). Other secondary endosymbionts' content and relative level would differ among the nymph and adult stages. For example, the relative abundance of Arsenophonus (22.89%) was higher than that of Wolbachia (4.52%) in the individual larva but lower in the adult stage where Arsenophonus accounted for 5.57% and Wolbachia increased to 11.58%. This fluctuation suggested a competition between Arsenophonus and Wolbachia in the growth of A. camelliae. This mutual exclusion relationship may be caused by its similar harbor in host cells and competition for limited resources as reported in other insects (Gottlieb et al., 2008; Qu, 2013). Furthermore, the substantial increase in Wolbachia level from the larva to adult stages in A. camelliae agreed with Wolbachia changes in Diaphorina citri (Meng et al., 2019) in our early report where Wolbachia symbiotic level kept increasing with the growth of tea green leafhopper (Zhang et al., 2022). These results from 16S rRNA sequences were confirmed by the qPCR method. The relative content was consistent in both ways, and all five investigated endosymbionts showed the same relative abundance level and changing tendency from the larva to adult stages in two analysis ways (Figures 2, 4). For example, the Portiera content kept at the highest level in both stages (Figure 4A), Arsenophonus dropped in adults (Figure 4B), and Wolbachia increased (Figure 4C). This indicated that this qPCR result was consistent with Illumina NovaSeq 6000, and the comparison was reliable.

Rifampicin was often applied to achieve aposymbiotic insects to check the change and influence of symbionts (Ahmed et al., 2010; Ren et al., 2021). We also employed rifampicin treatment to perform the symbiotic reduction in A. camelliae. We failed to selectively remove secondary symbionts without affecting the primary symbiont in A. camelliae. Some articles reported success in selectively removing the secondary symbiont Hamiltonella or Wolbachia in B. tabaci, using the same antibiotic rifampicin without affecting the primary symbiont Portiera (Xue et al., 2012; Su et al., 2015). Our results indicated that rifampicin could sufficiently reduce all investigated symbiotic bacteria of A. camelliae. Furthermore, Portiera was greatly reduced in rif-treatment but inhibited to less extent compared to secondary symbiotic bacteria changes. This phenomenon was also reported in Bemisia caused by rifampicin treatment (Shan et al., 2016; Zhao et al., 2020).

The age–stage two-sex life table can help researchers systematically understand the impact of removing symbiotic bacteria on the survival rate, development rate, and reproduction rate of insect populations, as well as the comprehensive impact of various factors on population growth (Chi et al., 2020). We first studied the role of symbiotic bacteria in A. camelliae by using the age–stage two-sex life table method. Our result indicated that rif-treatment markedly affected their biological parameters including the net reproductive rate (R₀), intrinsic rate of increase (r), and finite rate of increase (λ) (Table 5). Among them, the intrinsic rate of increase (r) is a basic ecological feature helpful to estimate the population growth potential (Roy et al., 2003). In our present result, (r) was decreased about 2-fold in the rifampicin treatment compared to the control. After symbiotic reduction, this indicated that the preoviposition period (including TPOP and APOP) was prolonged, and the adult longevity was shortened. Moreover, the control group had longer life expectancy and age–stage-specific survival rates in comparison to the rifampicin treatment. These changed biological traits negatively affected the development of A. camelliae, ultimately resulting in the reduction of the offspring population. The present results showed that aposymbiotic action had a clear unfavorable influence on A. camelliae, which is highly consistent with early reports (Karamipour et al., 2021). For example, bacterial symbiont elimination had negative effects on nymphal development and adult emergence of Brachynema germari, Acrosternum heegeri, and Acrosternum arabicum (Kashkouli et al., 2019) and adverse effects on life history traits of Graphosoma lineatum (Karamipour et al., 2021).

Symbiotic bacteria can help host insects degrade nutrient macromolecules in food and convert them into small molecules that can be directly used to provide missing nutrients for host insects. Endosymbiotic bacteria play an important role in the nutrition utilization of host insects (Frago et al., 2012). Luan et al. (2015) proved that Portiera, the primary symbiotic bacteria of Bemisia tabaci, was mainly involved in the nutrient metabolism of the host and could provide essential amino acids for the host. Previous studies have shown that Portiera may help whitefly improve their performance, and Portiera has a close nutritional relationship with their host during host plant acclimation (Hu and Tsai, 2020). The removal of Arsenophonus increased the developmental time of the immature stages and reduced the values of different life history parameters in Ommatissus lybicus (Karimi et al., 2019). We simply illustrated the function of symbiotic bacteria of A. camelliae by metagenomic analysis served by PICRUSt (Supplementary Figure 2D), which allows inference of the functional profile of the symbiotic bacterial consortia. It suggested that metabolism is highly responsible for the relative abundance of function. Whether the effect of symbiotic bacteria of A. camelliae is related to the synthesis of amino acids or other respect needs further research. It was needed to mention that rifampicin is broad spectrum and resulted in sufficient changes in symbiont titers of A. camelliae and downstream severe fitness defects, including elevated nymphal mortality and reduced population growth parameters. These changes in the population growth may be caused by the collaboration of all consortia of a specific bacterium. However, each specific bacterium and its function could not be specified in this present way. Though it has been reported that rif could specifically reduce Buchnera without affecting other bacterial compositions of Aphis gossypii (Ayoubi et al., 2020). Classification of the specific bacterium may be realized by the combination of sterilization and infection. On the other hand, this biological change of A. camelliae might not only involve bacterial clearance but also other changes in the microenvironment of the host.

Taken together, we sequenced the A. camelliae 16S rRNA gene through the Illumina NovaSeq 6000 platform, which directly revealed the structure of the bacterial community in the larva and adult stages and proved that Candidatus Portiera, Arsenophonus, Wolbachia, Rickettsia, and Pseudomonas genera of Proteobacteria dominated the whole life cycle of this whitefly. In addition, we confirmed that the rifampicin treatment can suppress the endosymbiont of A. camelliae and cause defective host fitness including a longer preadult stage in the offspring generation, a lower survival rate, decreased intrinsic rate of increase (r), and net reproductive rate (R₀).

The present study indicated that endosymbionts played an important role in the normal development and growth of A. camelliae. Regarding the applied aspect of Camellia spiny whitefly control, the rapid reduction in the primary and secondary symbionts and consequent death of the whitefly hosts indicate that antibiotics or agents targeted on these bacteria may possess the properties of rational insecticides to be used as part of a whitefly management program.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, PRJNA910681.

YT and BG conducted the experiments. YT and JW designed and performed the experiments. YT, QZ, and CL analyzed the data. LJ and XZ conceived and supervised the project. XZ and YT wrote the manuscript. All authors have read and agreed to the published version of the manuscript.