Performance of algorithm on defined media and SY-media datasets.

<p>Datasets contain 8 vial images. A Leave-one-out cross-validation strategy was performed for each vial (7 in and 1 out). Standard error was generated from n = 5 trials (statistical replicates). QuantiFly Accuracy represents the baseline algorithm with the bias correction.</p><p>Performance of algorithm on defined media and SY-media datasets.</p

The relationship between blue label uptake and observed feeding events did not change for flies of advancing age.

<p>Circles represent measurements of blue label uptake after 30 minutes of feeding and the proportion of feeding events observed during this period. One circle represents one vial containing 5 flies. Experiments were conducted with mated Dahomey females. Assays occurred at 4 different ages: on days 7, 21, 35 and 50 after eclosion. Each assay used 60 flies (12 vials) that were taken from a population that began with 500 individuals. Solid lines represent the significant (<i>P</i><0.0001) V/O relationship with a gradient coefficient of 160.36 (S.E. = 31.39) and intercept of 2.89 (S.E. = 3.45), dashed lines represent the line of best fit for each age class.</p

Schematic illustration of training and evaluation modes of QuantiFly software.

<p>(Left) Training mode: steps required to train a QuantiFly model to recognise eggs in an image scene. (Right) Evaluation mode: steps involved with evaluating a bulk number of images with a pre-trained model. Blue circles depict points at which a user must provide information to the system, either specifying input/output locations of files or through labelling eggs in images.</p

Measurements of blue label uptake after 30 minutes of feeding and the proportion of feeding events observed during this period, where one circle represents one vial containing 5 flies.

<p>Trend lines represent the relationship between the volume of food ingested and the observed proportion of flies feeding (V/O) described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006063#pone-0006063-t001" target="_blank">Table 1</a>. Dashed lines represent open circles. All flies were female unless stated, were 7 days old and were allowed to mate for 48 hours after eclosion (NF = the number of flies per condition, NV = the number of vials per condition). (a) A linear (V/O) relationship existed in mated Dahomey females (NF = 210, NV = 42). (b) The V/O relationships of mated Dahomey males and females did not differ significantly, although females were found to have fed at a greater frequency than males during the 30 minutes (NF = 200, NV = 40). The gradient for males did not differ significantly from that for females but had a lower intercept. (c) DR fed and full fed Dahomey females shared the same V/O relationship and no difference in feeding between dietary conditions was found with the combined assay (NF = 75, NV = 15). (d) The V/O relationship was the same in <i>chico</i>¹ heterozygotes and in the Dahomey control. No difference in feeding between genotypes was found with the combined assay (NF = 90, NV = 18). (e) The V/O relationship was the same in <i>takeout </i>¹ and in Canton-S females, even though <i>takeout </i>¹ flies were found to feed at a higher frequency than Canton-S controls (NF = 60, NV = 12). (f) Both <i>ovo</i>^D1 and <i>white</i>^Dahomey females had a positive V/O relationship, but <i>ovo</i>^D1 flies had a significantly greater gradient and intercept, and therefore increased the volume of food ingested per proboscis-extension more quickly than <i>white</i>^Dahomey females (NF = 200, NV = 40).</p

A linear relationship was tested between blue dye accumulations and feeding frequency using ANOVA in linear mixed effects model.

<p>The <i>P</i> value of the interaction terms is also displayed, which indicated whether the regression coefficients differ between comparative conditions (NF = no. of flies per condition and NV = no. of vials per condition).</p>a<p>These assays were not repeated on different trial dates. The statistical analysis was therefore only on fixed effects, i.e., a regression analysis.</p>b<p>These standard errors are for the differences in the intercepts.</p

Typical vial images illustrating varying lighting conditions and irregularities in media surface.

<p>Original vial image (A) and egg density representation from QuantiFly (B) for transparent defined medium (DM). Original vial image (C) and egg density representation (D) for opaque sugar/yeast (SY) medium. Red arrows highlight artefacts present in images: (1) clumped eggs; (2) marks on vial base; (3) bubble artefacts in media; (4) specular reflection from vial surface, and; (5) clumped eggs on food surface. Colour bar represents pixel density estimate of an egg.</p

The proportion of time spent feeding for DR (open circles) and full fed (FF) flies (closed circles) on different days of their lifespan.

<p>Survivorship curves are indicated with a solid grey line (DR) and a solid black line (FF) flies. Median lifespan: DR = 70 days, FF = 65 days. Proboscis-extension assays used 150 flies (30 vials) per condition. Flies were maintained in populations that began with 1500 individuals per condition (error bars = S.D.).</p

The proportion of time spent feeding during a proboscis-extension assay for DR (open circle) and fully fed (closed circle) once-mated 14-day old females.

<p>Flies were maintained on different diets throughout their lifespan. DR females did not differ from fully fed females in feeding frequency. The assay began immediately when the observer arrived. Note the lower proportion of flies feeding during the first 30 minutes of the assay, which may relate to the appearance of the observer in the room (NF = 100; NV = 20).</p

Comparison of QuantiFly performance when compared to human counter.

<p>Digital images were captured for four nutritionally different transparent media (A; C1-C4) and 4 different opaque media (B; D1-4). Estimates of the eggs in each vial were compared for the following methods: automated counts from QuantiFly algorithm; manual counts from a human and a digital on-screen ground-truth count (grey). (C) Image of opaque media vial with densely clustered eggs, red arrow 2 shows region with high-level of clustering. Error bars represent standard error of differences in vial densities in each condition (C1-4, n = 8; D1-4, n = 5 vials per condition).</p

Performance of QuantiFly on transparent and opaque media compared to human manual counts and digital ground-truth counts for each dataset.

<p>Q:QuantiFly prediction, M: Manual human counts, D: Digital ground-truth counts. Pairwise: Tukeys pairwise comparison; Fold Difference; the fold difference between counts; Correlation, Pearsons correlation coefficient for each comparison.</p><p>Performance of QuantiFly on transparent and opaque media compared to human manual counts and digital ground-truth counts for each dataset.</p