Diversity and associations between Drosophilidae (Diptera) species and Basidiomycetes in a Neotropical forest

ABSTRACT Drosophilidae is one of the most representative families of insects that occurs in fungal fruiting bodies of Basidiomycetes; however, the diversity and community structure of mycophagous Drosophilidae in the Neotropical region is poorly known. The aims of the present study were to describe the diversity of mycophagous Drosophilidae and to investigate its colonization of fungal hosts in a forest of southern Brazil. From 120 fungal samples (patches of mushrooms) of 17 Basidiomycetes genera, flies were recorded emerging from 70 samples and collected in adult stages of 25 fungal samples, for a total of 4897 drosophilids belonging to 31 species and 5 genera. Drosophila Fallén was the most species-rich genus, whereas Hirtodrosophila Duda was the dominant genus. Studies performed in the Holarctic region indicate that mycophagous drosophilid have generalist habits; however, our results showed that most drosophilids use fewer than two fungal hosts, and most species of Hirtodrosophila and Leucophenga were restricted to abundant fungal species, suggesting a specialization for these resources. The most specialized fauna emerged from Auricularia, which was the most frequent fungal genus in our collection, and this result supports the assumption that specialization depends on the availability of fungal resources over time

Reproductive diapause incidence on <i>D</i>. <i>americana</i> strains from north, center and south of the distribution.

<p>Reproductive diapause incidence on <i>D</i>. <i>americana</i> strains from north, center and south of the distribution.</p

Geographical origin and diapause incidence of different populations of <i>D</i>. <i>americana</i> across the USA.

<p>Diapause frequencies are represented by the color portion of the circular graphics close to the geographical coordinates and collection sites (O–Fremont, Nebraska, HI–Howell Island, Missouri; W–Lake Wappapelo, Missouri; RB–Pearl River, Mississippi; CB–Corney Bayou, Louisiana; and CI&SF–Cat Island, Saint Francisville, Louisiana) of the strains phenotyped (A). Diapause frequencies of each strain were used for the linear regression with the latitude of origin, and are presented by the average ± standard error of the mean (S.E.M) for each population. The slope of the curve represents the increase on diapause incidence with latitude and the R² value, the amount of variation on this phenotype explained by latitude (69.3%) (B).</p

<i>ActinD1</i> expression level can be used as a marker of ontogenetic delay, which results in extended LS, but it is not a marker of biological age.

<p>Differences in <i>ActinD1</i> gene expression levels were determined between 28 days old diapausing females (O53-D), 15 days old (O53-15d) and control females (O53-C) reared for 28 days under non-diapause inducing conditions (12L:12D at 25°C), as well as between 28 days old non-diapausing females (CB05.08-ND), 20 days old (CB05.08-20d) and controls (CB05.08-C) reared for 28 days under non-diapause inducing conditions (12L:12D at 25°C). The reference gene <i>RpL32</i> was used to normalize the expression values. Three biological replicates were used for each sample and the averages of the log-transformed values of 2^-∆CT are presented with their respective S.E.M.. A two-tailed Student’s <i>t</i> test assuming equal variances was used to address if the averages of normalized <i>ActinD1</i> expression levels of the different samples and treatments are significantly different (n.s. P > 0.05; * 0.05 > P > 0.01; ** 0.01 > P > 0.001; *** P < 0.001).</p

Lifespan (in days) summary statistics for diapausing (O53-D) and non-diapausing (CB05.08-ND) females reared under diapause inducing conditions (10L:14D at 11°C) for 28 days and then transferred into non-diapause inducing conditions (12L:12D at 25°C) and controls (C) reared exclusively under non-diapause inducing conditions.

<p>Lifespan (in days) summary statistics for diapausing (O53-D) and non-diapausing (CB05.08-ND) females reared under diapause inducing conditions (10L:14D at 11°C) for 28 days and then transferred into non-diapause inducing conditions (12L:12D at 25°C) and controls (C) reared exclusively under non-diapause inducing conditions.</p

The phenotype of <i>D</i>. <i>americana</i> reproductive diapause.

<p><i>Drosophila americana</i> 28 days-old ovaries of female flies reared since eclosion under non-diapausing conditions (12L:12D at 25°C, A), and reared since eclosion under diapause-inducing conditions (10L:14D at 11°C) showing non-diapause (B) or diapause (C) phenotypes. The non-diapausing (B’) and diapausing ovaries (C’) are also shown with higher magnifications. A scale of 500μm is provided in all pictures.</p

Diapause inducing conditions increase LS, which is more pronounced in diapausing females when compared with non-diapausing ones.

<p>Survival curves throughout time are shown for <i>D</i>. <i>americana</i> strains O53 (diapausing) and CB05.08 (non-diapausing) reared under non-diapause inducing conditions (12L:12D at 25°C; controls) and at the same conditions after being reared for 28 days under diapause inducing conditions (10L:14D at 11°C; O53-D (11°C+25°C) and CB05.08-ND (11°C+25°C)). Additional curves are presented for the same O53-D and CB05.08-ND females, but with LS measured starting from the day they were transferred into non-diapause inducing conditions (25°C).</p

<i>ActinD1</i> expression levels are highly correlated with LS expectancy.

<p>Fold changes in <i>ActinD1</i> expression levels obtained for 28 days old controls (O53-C and CB05.08-C), as well as for 28 days old diapausing (O53-D) and non-diapausing females (CB05.08-ND) were plotted against their respective LS values obtained under 12L:12D at 25°C (excluding the first 28 days). Three biological replicates were used for each sample, and the individual log-transformed expression values were used for the linear regression. The averages of the log-transformed values of 2^-∆CT are presented with their respective S.E.M.. The slope of the curve represents the increase in <i>ActinD1</i> expression levels with increased LS expectancy, and the R² value the amount of variation in expression levels explained by LS (94%).</p