The microbiomes associated with insects are important in mediating host health and fitness. In recent years, numerous studies have explored the microbial diversity and variations across different developmental stages in insects, particularly for pests, including Bactrocera dorsalis16, Monochamus alternatus17, and Zeugodacus tau18. Previously, bacterial communities were investigated using inefficient, low-throughput culture-based or conventional molecular methods19,20, inevitably underestimating the microbial abundance. The advancements in sequencing technology have inspired more research on insect microbial communities, thereby enriching the information on the microbiome of insects. However, a comprehensive understanding of the C. megacephala pupal stage microbiome remains unclear. Therefore, this paper presents a study of the diversity and dynamics of bacteria in the pupal stage of C. megacephala using third-generation sequencing of bacterial 16S rRNA. The results provide a better understanding of the C. megacephala microbiome.
This annotation results demonstrate that the bacteria in the pupal stage of C. megacephala are rich and diverse, but the diversity is indiscrete. At the phylum level, Proteobacteria, Firmicutes, and Bacteroidetes were the three predominant phyla, similar to the observation from the housefly Musca domestica21, possibly owing to a semblable ecological niche. The bacterial community analysis identified Clostridia and Gammaproteobacteria as the two predominant bacterial classes in the pupal stage of C. megacephala with ~ 30% relative abundances. However, another study of the gut bacteria across the lifecycle of C. megacephala showed Gammaproteobacteria as the dominant class with over 60% relative abundance. These results suggest that Clostridia may be from other C. megacephala tissues apart from the gut.
Compared with the previous results about C. megacephala bacterial communities that were determined using culture-based or conventional molecular methods, the microbial diversity was much higher in this study using third-generation sequencing technology22. However, we cannot identify some bacteria to the species level, such as Klebsiella pneumoniae and Aeromonas hydrophila23, so culture-based and conventional molecular methods are also important.
Ignatzschineria indica and Wolbachia endosymbiont were the two predominant species in the bacterial communities in the C. megacephala pupal stage. Ignatzschineria indica is a Gram-negative bacterium commonly associated with maggot infestation and myiasis, a probable marker for myiasis diagnosis24,25. Wolbachia are intracellular symbiotic bacteria widely distributed in the reproductive tissues of arthropods. They cause reproductive alterations in their hosts, such as cytoplasmic incompatibility (CI)26, feminization27, killing males28, and inducing parthenogenesis (PI)29. Wolbachia increases the resistance to arbovirus infection, resulting in decreased virus transmission. The reproductive regulation of Wolbachia on target organisms may be important in future biological prevention and pest control. Since Wolbachia causes CI, Wolbachia-infected populations can be established and released to reduce to the environment to reduce the reproductive potential of harmful target insect populations. Modified Wolbachia that harbor anti-parasitic or anti-viral genes can be adopted to control virus transmission in insects carrying viruses30.
However, few studies have reported that Ignatzschineria and Wolbachia can coexist in an individual insect, despite their status as common bacterial genera. Several possibilities may explain this analytical discrepancy. Firstly, in this study, Spearman’s rank correlation between Wolbachia and Ignatzschineria showed a negative correlation, suggesting a competitive relationship between Wolbachia and Ignatzschineria. Secondly, the previous investigations of bacterial communities applied inefficient, low throughput culture-based or conventional molecular methods, potentially generating incomplete results. Finally, numerous studies have established that microbial communities differ between insect populations because of different sampling techniques and procedures31. This study analyzed C. megacephala sampled from a laboratory population reared with pork for five years. Nevertheless, the significant decrease in the relative abundance of Wolbachia observed at the end of the pupal development is unsolved, thus, required further studies.
Traditionally, the most common method for pest control is by chemical pesticides. However, the excessive use of chemical pesticides causes the rapid build-up of pesticide resistance and environmental pollution. Therefore, it is urgent to develop biological control methods for pests. Nasonia vitripennis (Walker), is an important parasitoid whose female wasp stings, injects venom, and lays eggs in different fly pupae, where parasitoid eggs, larvae, pupae, and early-stage adults develop. N. vitripennis lives in species of the family Calliphoridae, Sarcophagidae, and Muscidae, where their larvae feed on fly pupae, allowing N. vitripennis to function as a biological agent to control the flies.
The microbial communities of fly species and N. vitripennis live in an enclosed environment, providing more opportunities for the N. vitripennis-fly communication. Therefore, the impacts of micro-communities of the fly hosts on N. vitripennis are worth studying, precisely at the pupal stage. Studies of different fly hosts and their corresponding N. vitripennis showed diverse core microbiota, and so other fly hosts shaped the bacterial diversity of their parasitic wasps32. In addition, parasitic wasps infected with Wolbachia produced more female offspring than uninfected ones, further emphasizing the need to improve biological prevention and control efficiency33. Therefore, a deliberate focus to study the micro-communities of different fly species at the pupal stage and the interaction between the fly species and N. vitripennis will guide the development and utilization of N. vitripennis as biological agents for the prevention and control of flies.
Approximately half of the bacteria identified at the species level in this study are pathogens or conditional pathogens (Supplementary Table S2), Escherichia coli, Providencia burhodogranariea, and Morganella morganii, among others. Another uncommon pathogenic bacterium, Erysipelothrix rhusiopathiae was also identified at the species level. E. rhusiopathiae is the etiological agent of swine erysipelas and causes economically important chicken, duck, and sheep diseases. Although E. rhusiopathiae primarily infects pigs, it also infects various domestic and wild mammals, including marine mammals, birds, and humans. Humans infected with E. rhusiopathiae develop large areas of red spots on their body. Severe E. rhusiopathiae infection causes endocarditis and septicemia, which have a 38% mortality rate34.
However, very few studies have focused on the insects that transmit E. rhusiopathiae35. Considering that the C. megacephala samples in this study were obtained from a laboratory population reared for five years, it is likely that the E. rhusiopathiae originated from infected pork and were transmitted to C. megacephala through feeding. Thus, disease-vector insects can infect and spread pathogens beyond their feeding activities, and disease-vector insects require more comprehensive prevention and control methods (“Supplementary information”).
In conclusion, this study comprehensively investigated the pupal stage microbiome of C. megacephala using third-generation sequencing to deepen the understanding of C. megacephala microbial communities on the whole. The study provides a basis for subsequent studies of biological control and the comprehensive utilization of C. megacephala. Future studies should focus on the transmission patterns and biological functions of these microbial species.
Source: Ecology - nature.com
