The analysis of temporal dynamics of the gut microbial community structure across the Galerucella life stages revealed a dynamic community which depends on whether females deposited a fecal string on the eggs or not. Whereas the microbial diversity was quite stable between life stages in fecal species, it was much more variable in non-fecal species. In particular, the diversity of dominant gut bacteria taxa in the non-fecal species peaked during the egg stage and then decreased strongly from egg to pupal stages (Fig. 2). We also found large differences in the microbial community between species (similar to10) and we specifically found several bacteria that occur consistently across life stages and that differed between the beetle hosts. For instance, bacteria such as Acinetobacter, Erwinia and Wolbachia were exclusively found in non-fecal species whereas Novosphingobium and Flavobacterium were more commonly found in fecal species (Fig. 3).
Although the microbial community composition may be strongly affected by metamorphosis in holometabolous insects19,20, we found multiple ASVs shared across life stages in both fecal and non-fecal species, which indicates a potential generational inheritance (Fig. 3). These data suggest vertical transmission mechanisms in both fecal and non-fecal species, even though the latter have no apparent transfer mechanism. In the fecal species, the fecal strings laid on the eggs would efficiently transfer microbial communities from mother to offspring, as these fecal strings are consumed by the newly hatched larvae. Presumably, microbes are similarly transferred in the non-fecal species by females through some other mechanism, such as coating the egg during the egg-laying process (cf. 3), but this mechanism is less efficient and likely reduces the relative dominance of transferred taxa relative to environmentally acquired taxa. Due to the high number of microbial cells in the fecal strings, there is a presumably a high dominance of transferred taxa in fecal species and a more stable microbial dynamic.
The colonization of gut microbial communities in offspring is obviously important and affect internal dynamics, a process that can be affected by secretions that parent insects add on the eggs (called pre-hatch care) or by females feeding offspring with bacteria (called full care)20. For instance, studies on burying beetles showed that larvae receiving pre-hatch care and full care were colonized with bacteria from the maternal gut, whereas larvae that did not receive such care picked up bacteria from the carcass20. In another insect species, the oriental fruit fly (Bactrocera dorsalis), bacteria (Citrobacter freundii) were found in deposited eggs despite not being detected in ovaries21. Furthermore, in bumblebee, core gut bacteria exist throughout the different development stage, which, in combination with observed similarities in microbial communities between generations, suggest vertical transmission of gut bacteria19. These examples indicate that endosymbionts could be vertically transmitted through smearing as the egg passes such as fecal strings. Transmission mode, especially vertical transmission, can also influence host dependence and host reproduction, and in return, influence niche colonization22,23.
In other species, symbionts can be transferred through elaborate mechanisms in the host reproductive system. For instance, in the rice weevil Sitophilus oryzae, the principal symbionts are associated with both primordial germ cells and the future bacteriome and are transferred to the next generation through the ovaries24. There may even be pseudovertical transmission as is the case between the entomopathogenic nematode Steinernema carpocapsae and its symbiont, Xenorhabdus nematophila, where the symbionts transmitted between nematode generations not only come from the parent (vertical) but also from co-occurring nematodes in their host25. The symbionts of Drosophila melanogaster even occur in the male germ line26, illustrating differences in the vertical transmission routes among species. We suggest that fecal strings is an additional transfer mechanism which has received limited interest in insect research. Our understanding of transmission mechanisms does not yet extend to their causes and consequences. However, figuring out the dynamic of gut microbial communities between fecal and non-fecal could direct our attention to crucial factors linked with mode of transmission.
The microbial diversity in the beetle guts were obviously large. ASVs belonging to Pseudomonas, Stenotrophomonas, Sphingobacterium, Acinetobacter, Serratia, Erwinia, Novosphingobium, Flavobacterium and Wolbachia were dominant bacteria taxa in different life stages of the five Galerucella species (Figs 1 and 3) but differently in different species. The effect from these consistently occurring bacteria on the host, and how differences in the microbial community differentially affect species, can only be speculated upon. In some cases, functions are known from other insect taxa and the list of common bacteria in Galerucella includes both those shown to benefit their host and those that are potentially pathogenic. For instance, Serratia dominated in non-fecal, but not fecal, species across life stages (Fig. 1), and this genus is known as gut symbionts that could infect many insect species27-31, but multiple Serratia species are also known as insect pathogens32. Other common symbionts in this study, such as Stenotrophomonas and Pseudomonas, have previously been found to depress plant defenses33,34 whereas Acinetobacter have been shown to degrade complex organic molecules35. In other cases, eventual functions are more unclear. Erwinia, which was commonly found in non-fecal species is mainly described as a phytobacterial pathogen, the causal agent of fire blight, and usually infect plants through insects such as aphids36,37.
Wolbachia was in our study only found in non-fecal species and is a well-known facultative symbiont in insects. They are often maternally inherited bacteria related with feminization, parthenogenesis and cytoplasmic incompatibility (CI)38,39. For Wolbachia to become established in a new host species, it must be horizontally acquired and vertically transmitted at a high rate40. In D. mauritiana, there are three possible ways that Wolbachia could transfer between population: as the neutral bacteria, as beneficial bacteria, or related to with the spindle apparatus of nuclei during division41. The vertical transmission of Wolbachia in Acari proved that Wolbachia-infected females transferred Wolbachia to their offspring regardless they mated with infected or uninfected males, but the offspring of Wolbachia-free females were always uninfected42. In Galerucella species, we find that non-fecal species possess a much higher level of Wolbachia than fecal species, almost exclusively (Figs. 1 & 4). This pattern could suggest that fecal strings possibly hinder Wolbachia transmission or establishment through the maintenance of some other beneficial microorganisms.
To conclude, our findings demonstrate that the dynamics of gut bacteria and the bacterial community are impacted by fecal strings across host life stages, with a very different microbiome shared between fecal and non-fecal species. Importantly, this work also suggests that different bacterial genera may be both positively and negatively related with fecal strings. This information suggests that understanding the temporal dynamics of gut microbial communities in both fecal and non-fecal species may help identify key drivers affecting the assembly of microbiomes and potentially how the microbial functions related to insect fitness. Nevertheless, we find that the gut microbiota structure both by phylogenetic perspective and environmental perspective, where the innovation of adding fecal strings to the egg could play a pivotal role in this particular beetle genus.