A qPCR assay was used to examine symbiont inocula within cocoons, as well as the relative growth dynamics of leech symbionts through embryonic development. We chose to analyze the proliferation of A. veronii and Ochrobactrum and Niabella spp., as these are the more prevalent symbionts, through host embryogenesis. qPCR was performed on cocoon albumen at ≤24 h PD, and individuals were extracted at 6, 17, 21, and 25 days PD using an iCycler iQ real-time PCR detection system (Bio-Rad, Hercules, CA). For each corresponding time point, measurements were collected from at least three samples. A. veronii gyrB (NCBI accession no. AY299320) and species-specific portions of the 16S rRNA gene were used for Niabella and Ochrobactrum species quantification, respectively. The H. verbana cytoplasmic actin gene (Hirudo Act1) was used for normalization of samples. The fluorometric intensity of SYBR green I dye (Bio-Rad, Hercules, CA) was used for gene quantification. The PCR amplification parameters, reagents, primer design criteria, and sequences used for quantification are summarized in Table .
Materials and amplification conditions used in qPCR of symbiontsa
Symbiont density is defined as the relative ratio of symbiont genes to host cell number. Internal standard curves were generated for each primer combination using the four above-mentioned amplicons, each cloned into pGEM-T vector (Promega, Madison, WI). Density estimates were obtained by comparison to a standard curve using Bio-Rad iCycler iQ multicolor real-time PCR data analysis software. Quantitative measurements were carried out using 96-well plates in duplicate. Replicates were averaged for each sample prior to the construction of relative copy number ratios. Negative controls were included in all amplification reactions. Values are represented as means (± standard errors of the means).
Data were analyzed using JMP 7.0 software (SAS Institute, Cary, NC). Analysis of variance and post hoc pairwise comparison of the means were performed to determine whether densities significantly differed between time points. The normality of density distributions was determined with a goodness-of-fit test. Symbiont densities were log transformed to satisfy normality.
The time after cocoon deposition was shown to significantly affect the density of the examined symbiont species (F = 56.1, P < 0.0001), suggesting oscillating population dynamics through host embryogenesis. At ≤24 h PD, the three symbiotic species are found at different abundances within the cocoon albumen, with A. veronii at a significantly lower density than the comparable abundances of Ochrobactrum and Niabella spp. (F = 9.38, P < 0.0001; results not shown). These differences in inoculum sizes within an individual cocoon may be a factor impacting the frequency of infected individuals or reflect a difference in the transmission of bladder symbionts rather than crop symbionts.
Correspondingly, these three symbiont species also displayed diverse growth patterns within individual cryptolarvae and juvenile leeches (Fig. ). The A. veronii
are found at higher concentrations during the onset of embryogenesis that decreases toward midembryogenesis, although statistical significance was obtained only for the former symbiont. This initial decrease is likely due to the more rapid increase in the host actin signal, as the leech cells proliferate during development. In contrast to the increase in density of Ochrobactrum
spp. during late embryonic development (i.e., juveniles at 21 versus 25 days PD), A. veronii
remained stagnant. Typically, A. veronii
is harbored at low levels within starved adult leeches (sometimes below the limit of detection), with its abundance rapidly increasing following the consumption of a blood meal (9
FIG. 3. Symbiont density dynamics through leech embryogenesis of Ochrobactrum sp. (A), A. veronii (B), and Niabella sp. (C). Each averaged ratio, for all three species, came from the same set of samples used for each time point. Values are represented by means, (more ...)
Intuitively, one assumes that extracellular digestive tract symbionts are acquired horizontally after hatching or birth. We present evidence that supports the vertical transmission of A. veronii
to its leech host. Our experiments reveal the dynamic nature of symbiont invasion through host development by microbial species in terms of inoculum size, time of establishment, and frequency of infection even by a relatively simple microbiota. Synchronization of host development with the establishment of particular members of the microbial community is likely important for niche accessibility, particularly given the microbial stratification observed within adult hosts (10
). The microbial stratification also suggests the presence of intimate interactions, not only host-microbe but also microbe-microbe mediated, which will affect the selective process, perhaps by promoting the recruitment and stabilization of heterospecifics. Competition or, contrastingly, cooperation between host-associated microbes could affect density, virulence, or transmission modes of cohabitating microorganisms. Future studies will explore the potential role of symbiont species in promoting host establishment by other microbial species.