Body size and symbiotic status influence gonad development in \u3cem\u3eAiptasia pallida\u3c/em\u3e anemones

Pale anemones (Aiptasia pallida) coexist with dinoflagellates (primarily Symbiodinium minutum) in a mutualistic relationship. The purpose of this study was to investigate the role of these symbionts in gonad development of anemone hosts. Symbiotic and aposymbiotic anemones were subjected to light cycles that induced gametogenesis. These anemones were then sampled weekly for nine weeks, and gonad development was analyzed histologically. Anemone size was measured as mean body column diameter, and oocytes or sperm follicles were counted for each anemone. Generalized linear models were used to evaluate the influence of body size and symbiotic status on whether gonads were present and on the number of oocytes or sperm follicles produced. Body size predicted whether gonads were present, with larger anemones being more likely than smaller anemones to develop gonads. Both body size and symbiotic status predicted gonad size, such that larger and symbiotic anemones produced more oocytes and sperm follicles than smaller and aposymbiotic anemones. Overall, only 22 % of aposymbiotic females produced oocytes, whereas 63 % of symbiotic females produced oocytes. Similarly, 6 % of aposymbiotic males produced sperm follicles, whereas 60 % of symbiotic males produced sperm follicles. Thus, while gonads were present in 62 % of symbiotic anemones, they were present in only 11 % of aposymbiotic anemones. These results indicate that dinoflagellate symbionts influence gonad development and thus sexual maturation in both female and male Aiptasia pallida anemones. This finding substantiates and expands our current understanding of the importance of symbionts in the development and physiology of cnidarian hosts

Methodological precision of in situ and in vitro algal density measurements in the model cnidarian, Exaiptasia diaphana

In cnidarian symbiosis research, studying algal uptake, maintenance, and expulsion typically requires quantification of algal density in host tissue. Multiple methods are used to measure algal density including in vitro cell counts of holobiont homogenate and in situ cell counts of tentacle clippings. The relative precision of both types of measurement has not previously been reported for the model cnidarian Exaiptasia diaphana in the fully symbiotic state. The objective of this study was to evaluate the precision of in vitro and in situ algal density measurement protocols using light, fluorescent, and confocal microscopy and an automated cell counter. In situ algal density was quantified as algal area fraction (%) using confocal images of tentacle clippings mounted on two types of slides. In vitro algal density of holobiont homogenate was quantified as algal cells/µl of holobiont homogenate using an automated cell counter and a hemocytometer viewed using light and fluorescent microscopy. Triplicate measurements of each method for ten anemones were collected and the coefficient of variation was calculated and compared across the ten anemones within each method. The algal density measurements were equally precise when they were obtained by quantifying in vitro cell counts using a hemocytometer and when they were obtained by quantifying in situ cell counts. While both light and fluorescent microscopy yielded similar measurement precision of in vitro cell counts, use of a fluorescent microscope was more efficient and convenient than use of a light microscope, and both methods required terminal sampling. Conversely, in situ methods required more sophisticated equipment (namely a confocal microscope) but involved non-terminal sampling. An automated cell counter was ineffective for in vitro quantification of algal density, although the potential utility of this technology warrants future attempts using a more robust algal cell purification process that could include filtering homogenate prior to analysis. This study demonstrated that in vitro and in situ methods yield estimates of algal density with comparable precision, which is information that researchers can use for future studies when making decisions about methodology

Bridging developmental boundaries: lifelong dietary patterns modulate life histories in a parthenogenetic insect

Determining the effects of lifelong intake patterns on performance is challenging for many species, primarily because of methodological constraints. Here, we used a parthenogenetic insect (Carausius morosus) to determine the effects of limited and unlimited food availability across multiple life-history stages. Using a parthenogen allowed us to quantify intake by juvenile and adult females and to evaluate the morphological, physiological, and life-history responses to intake, all without the confounding influences of pair-housing, mating, and male behavior. In our study, growth rate prior to reproductive maturity was positively correlated with both adult and reproductive lifespans but negatively correlated with total lifespan. Food limitation had opposing effects on lifespan depending on when it was imposed, as it protracted development in juveniles but hastened death in adults. Food limitation also constrained reproduction regardless of when food was limited, although decreased fecundity was especially pronounced in individuals that were food-limited as late juveniles and adults. Additional carry-over effects of juvenile food limitation included smaller adult size and decreased body condition at the adult molt, but these effects were largely mitigated in insects that were switched to ad libitum feeding as late juveniles. Our data provide little support for the existence of a trade-off between longevity and fecundity, perhaps because these functions were fueled by different nutrient pools. However, insects that experienced a switch to the limited diet at reproductive maturity seem to have fueled egg production by drawing down body stores, thus providing some evidence for a life-history trade-off. Our results provide important insights into the effects of food limitation and indicate that performance is modulated by intake both within and across life-history stages

Age and size at each life-history transition.

<p>Points represent means (± standard errors) at the end of each instar, at first oviposition, and at death. U = unlimited access to food, L = limited access to food. Sample sizes: UUU <i>n</i> = 13, ULL <i>n</i> = 13, UUL <i>n</i> = 13, LLL <i>n</i> = 19 juveniles and 7 adults, LUU <i>n</i> = 12. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111654#pone.0111654.s014" target="_blank">Table S3</a> for statistical results for size and age at each point.</p

Stepwise multiple linear regression models predicting fecundity (models A1 to A5), initial egg output (models B1 to B4), adult lifespan (models C1 and C2), reproductive lifespan (models D1 and D2), and total lifespan (model E1).

<p>Notes: Abbreviations: Fecund = fecundity (number of eggs), 6d Eggs = early egg production (number of eggs oviposited during the first six days of the reproductive lifespan), Ad Life = adult lifespan (days), RL = reproductive lifespan (days), Tot Life = total lifespan (days), Int RL = total dry matter intake (g) during the reproductive lifespan, Nit 6-Ov = nitrogen (g) assimilated between the beginning of the sixth instar and first oviposition, SGR 5 = specific growth rate for body mass (per day) during the fifth instar, Ad SGR = specific growth rate for body mass (per day) during the adult stage prior to first oviposition, SGR 3 = specific growth rate for body mass (per day) during the third instar, SGR 1 = specific growth rate for body mass (per day) during the first instar, Len Ov = body length (mm) at first oviposition, MS Int 3 = average mass-specific intake (g/g) during the third instar, MS Int RL = average mass-specific intake (g/g) during the reproductive lifespan, MS Int Ad = average mass-specific intake (g/g) during adult stage prior to first oviposition, Nit 1-Ov = nitrogen (g) assimilated between the beginning of the first instar and first oviposition, Nit 5 = nitrogen (g) assimilated during the fifth instar, MR 4 = mass-specific metabolic rate (µL/g/hr) in the fourth instar. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111654#pone.0111654.s012" target="_blank">Table S1</a> for the independent variables tested with each dependent variable. Significant independent variables are listed in the order in which they were selected by the models. All models are significant at <i>p</i><0.0001 with <i>n</i> = 58 insects.</p><p>Stepwise multiple linear regression models predicting fecundity (models A1 to A5), initial egg output (models B1 to B4), adult lifespan (models C1 and C2), reproductive lifespan (models D1 and D2), and total lifespan (model E1).</p

Treatment groups and mass-specific intake.

<p>(A) Experimental design. Lifespans are represented by horizontal bars divided into six instars and an adult stage. Time is not to scale, and differences in timing of life-history transitions between groups are not graphically presented. Vertical lines in juvenile stages denote ecdyses. White bars represent life stages when food was offered ad libitum (U, unlimited access to food). Shaded bars represent life stages when food was limited (L) to 60% of the amount of food consumed by insects in group UUU on a percent body mass basis. Because survival to first oviposition was low for insects that were food-limited for the duration of juvenile development, we were unable to test the effects of a diet switch from L to U at first oviposition (LLU). Sample sizes reflect the number of individuals present in each treatment group at the beginning of the trial. (B) Daily mass-specific dry matter intake (g/g/day). Curves were constructed by scaling the duration of each stage for each insect to the average duration of that stage for each treatment group and fitting a loess smoothing function to these data. Points where mass-specific intake declined to zero correspond to ecdyses. The first six time intervals represent juvenile stages; the final time interval represents the adult stage. Arrowheads denote the average age at first oviposition. Mass-specific intake for UUU insects declined after first oviposition. The amount of food offered to food-limited adults after first oviposition was decreased proportionally to match this decline. Sample sizes: UUU <i>n</i> = 13, ULL <i>n</i> = 13, UUL <i>n</i> = 13, LLL <i>n</i> = 19 juveniles and 7 adults, LUU <i>n</i> = 12.</p

Reproductive performance.

<p>(A) Cumulative fecundity of insects in each of five treatment groups. The x-axis represents days of the reproductive lifespan. Each curve terminates at a point corresponding to the mean duration (± standard error) of reproductive activity and the mean fecundity (± standard error) for each group. Curves were constructed by scaling the reproductive lifespan of each insect to the mean reproductive lifespan for that group, determining the mean cumulative fecundity of all insects in that group on each day of the scaled reproductive lifespan, and fitting a smooth spline (<i>df</i> = 7) to the resulting means. U = unlimited access to food, L = limited access to food. Sample sizes: UUU <i>n</i> = 13, ULL <i>n</i> = 13, UUL <i>n</i> = 13, LLL <i>n</i> = 7, LUU <i>n</i> = 12. Different letters to the right of each point indicate significantly different means for fecundity (a, b, and c) and reproductive lifespan (w, x, y, and z) among treatment groups. (B) Metrics of egg production. Early egg output (white squares) was measured as the number of eggs oviposited during the first six days of the reproductive lifespan, and mean egg mass (black diamonds) was measured in mg (means ± standard errors). U = unlimited access to food, L = limited access to food. Sample sizes: UUU <i>n</i> = 13, ULL <i>n</i> = 13, UUL <i>n</i> = 13, LLL <i>n</i> = 7, LUU <i>n</i> = 12. Different letters indicate significantly different means for early egg output (a, b, and c) and mean egg mass (x, y, and z) among treatment groups.</p

Kaplan-Meier survivorship curves for (A) the entire lifespan and (B) the adult lifespan.

<p>Abbreviations: U = unlimited access to food, L = limited access to food. For A, sample sizes are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111654#pone-0111654-g004" target="_blank">Figure 4</a>, including insects that died prior to the adult molt (UUU <i>n</i> = 15, ULL <i>n</i> = 15, UUL <i>n</i> = 14, LLL <i>n</i> = 28, LUU <i>n</i> = 14). For B, only insects that survived to adulthood are included, and sample sizes are UUU <i>n</i> = 13, ULL <i>n</i> = 13, UUL <i>n</i> = 13, LLL <i>n</i> = 19 (7 of which oviposited), LUU <i>n</i> = 12. For graph A, pairwise log-rank tests indicated that all groups except LLL and LUU differed significantly in longevity. For graph B, UUU and LUU insects had significantly enhanced adult longevity compared to ULL, UUL, and LLL insects.</p

Event history diagram for individual insects maintained on five diet treatments.

<p>Each horizontal line represents the lifespan of one individual, with insects in each group arranged in order (top to bottom within a treatment group) from shortest to longest lifespan. U = unlimited access to food, L = limited access to food, RL = reproductive lifespan. The adult molt is indicated by a vertical black line. Data for insects that died during the juvenile stages are included in this diagram but were not included in any analyses except for survivorship curves (Fig. 5). Sample sizes: UUU <i>n</i> = 15, ULL <i>n</i> = 15, UUL <i>n</i> = 14, LLL <i>n</i> = 28, LUU <i>n</i> = 14.</p