Potential phenotypic plasticity within Simulium nigrimanum Macquart, 1838 (Diptera: Simuliidae) larvae

Black fly larvae (Diptera: Simuliidae) are suspension filter-feeders which strongly depend on water velocity for proper feeding. Black fly species feature different microhabitat preferences. Studies of Holarctic black fly larvae revealed their phenotypic plasticity in response to water current velocity variation, but such studies have been rarely undertaken with Neotropical black flies. The current work presents results on the phenotypic plasticity of the black fly species Simulium nigrimanum Macquart. Twelve last instar larvae, sampled from the Brazilian Cerrado, were photographed under a stereoscopic microscope and measured using the CMEIAS Image tool software. Linear regressions with water velocity as the independent variable were performed, indicating that while body size and anal disk diameter correlated positively with water velocity, labral fan length correlated negatively. The observed relationships between water velocity and labral fan length and anal disk diameter were consistent with the literature, while the pattern of body size variation partially corroborated previous studies. The present work results suggest that potential phenotypic plasticity can be observed in black fly larvae within one population distributed in different microhabitats of the same stream section, as opposed to previous reports implying that such variation is only found among population from different streams.


Introduction
Although black flies (Diptera: Simuliidae) are a cosmopolitan insect family with world wide geographic range, their local occurrence is limited by suitable microhabitat characteristics allowing immature filter feeding black fly larvae to breed develop [1]. Black fly populations are regulated by biotic and abiotic factors, the former being competition, predation, and disease [2,3]; and the latter involving to microhabitat features such as substrate and water velocity [4,5,6,7]. Black flies are key organisms in lotic systems, regarded as ecosystem engineers. This is due to the silk their larvae produce essentially altering the microhabitat enabling other organisms to fixate more easily in the substrate. The water velocity influences their ability to capture organic matter in suspension in the water column [7], and due to their ability to convert smaller particles of organic matter into larger nutritive fecal pellets of organic matter because of their low digestive efficiency they are considered key organisms in the boreal biome [7,8], and also the adults of some species are important disease vectors [9,10,11].
Black fly larvae usually reach their pupal stage on the same microhabitat they spent their last instar larval stage [7].
Ecological theory suggests that niche partitioning through differential use of resources is a key factor to diversity, as it promotes the local coexistence of ecologically similar black fly species [12]. This coexistence can be reached due to differential adaptations to microhabitat aspects; Nearctic and Palearctic black fly larvae are known to show different labral fan morphologies in relation to species level preferences [13,14]. Likewise, body size is strongly related to water velocity and food availability [15,16]. Even though these morphological relations are often studied in Holarctic black fly larvae, in their Neotropical relatives, these phenomena have rarely been approached [17].
In addition to its role as potential vector of the onchocerciasis, literature has suggested that the black fly species Simulium nigrimanum Macquart 1838 is potentially related to the skin condition known as Pemphigus Foliaceus [18,19]. This makes the investigation of the bionomic and ecological traits of this species extremely relevant to public health. Simulium nigrimanum is widely distributed in South America [18,19] and their larvae live associated to rocky substrates on small streams with dense canopy cover [11]. In this paper we investigate the relationships among S. nigrimanum morphological traits and water velocity; evaluating if these traits are correlated to this abiotic factor and among themselves.

Study area
The larva used in this study were originally sampled in from the Córrego do Mato (S12°39 0 33:0 00 , W48°18 0 27:3 00 ), which is a small stream shaded by a dense canopy cover located in the Brazilian Cerrado from the state of Tocantins in northern Brazil [11]. The Cerrado is a savanna biome which presents a typical vegetation consisting of grasses and trees that do not provide a close canopy cover, due to the small size and low density of its trees, with the exception of the gallery forests, which are the riparian vegetation of this biome.

Larvae sampling and identification
Black fly larvae of later instars were sampled by hand during six campaigns on alternate months from October 2004 to August 2005. A 15 m stream section had 15 quadrats (30 cm 30 cm) sampled during each campaign.
Larvae were fixed in 70 % ethanol and later sorted according to morphotype in the laboratory. Last instar specimens were dissected and identified using the taxonomic literature [20,21] and direct comparison with pupae collected in the sites and with the material deposited at the Laboratório de Simulídeos e Oncocercose/Instituto Oswaldo Cruz (LSO-IOC).
Twelve last instar individuals of S. nigrimanum were then separated for use in the present study. Larvae were then photographed in a stereoscopic microscope equipped with a digital camera and later measured with the use of CMEIAS software [22]. The decision to analyze only last instar larvae was made, compromising total sample size, to avoid any potential bias from measuring different stage larvae, which may still be prone to drift and morphological structure growth.

Larval measurements and statistical analyses
Each larva had its anal disk diameter, body length, and labral fan length measured according to the methodology described in Figueiró et al. [17] (Fig. 1). Whereas in this aforementioned study three different populations of Simulium subpallium Lutz 1909 from different lotic systems had their morphological traits compared, the present study assessed phenotypic plasticity within the same S. nigrimanum population investigating the patterns of variation within a single lotic system. All statistics and graphics were performed using PAST 4.3. These measures and the water velocities had their normality investigated using the Lilliefors test, and as all distributions were normal correlations among them and the water velocity were determined using the Pearson correlation coefficient. These morphological measures then were used as dependent variables in linear regressions in which the independent variable was the water velocity, which was measured in the field through the head rod method [23,11].

Results and Discussion
A total of 430 S. nigrimanum larvae were collected, from which 15 were last instar larvae. Three of these 15 specimens were dissected in order to confirm species identity and the remaining larvae were measured ( Table 1). All variables were significantly correlated ( Table 2). Linear regressions indicated that water velocity correlated positively with anal disk diameter (p < 0:01, r 2 D 0:73) and body length (p < 0:05, r 2 D 0:42) and negatively with labral fan length (p < 0:01, r 2 D 0:69) (Fig. 2).   The labral fan patterns were consistent with previous studies in the literature on Holarctic species, with smaller labral fans selected in faster water flow velocities due to the decrease in the cost of drag [15,16,24], however the observed body size variation pattern differed from that in these studies in which small larvae were observed in streams with the highest water flow velocities [15].

Specimens velocity Anal disc diameter body length labral fan length
This divergence of the pattern of body size correlation to water velocity can be attributed to the range of water velocities sampled (0:82 m s 1 to 1:25 m s 1 ). Zhang & Malmqvist [15] found that maximum body sizes were observed at moderate velocities with size decaying at faster velocities or, as suggested by Lucas & Hunter [24], due to food availability. The abundance of food can also influence morphological traits in the case of optimal conditions for this species being found on faster flows.
The anal disk diameter was consistent with the pattern observed for S, subpallidum Lutz, 1910 larvae [17], with larger diameters observed at higher water flow velocities, probably due to these diameters accommodating more hooks or more robust hook structures that allow larvae to resist drag.

Conclusions
These results suggest that potential phenotypic plasticity can be observed in black fly larvae within one population distributed through different microhabitats of a stream section, and this also represents the first data regarding S. nigrimanum phenotypic plasticity in response to microhabitat features. This data allows for a better comprehension of the spatial distribution of S. nigrimanum, which is considered a potential vector of the Onchocerciasis. The patterns observed in this study also suggest that although some of the trends observed in Neartic and Paleartic black fly larvae may be similar to those of Neotropical black flies, possibly some traits show markedly different patterns in the Neotropics.

Acknowledgements
The present work was funded via a Postdoctoral grant to Figueiró R. by Programa de Pósgraduação em Biodiversidade e Saúde / Fundação Oswaldo Cruz at Laboratório de Simulídeos e Oncocercose. The authors thank CNPq, FAPERJ, and FIOCRUZ for the financial support and the staff of Laboratório de Simulídeos e Oncocercose for all their support particularly with data collection and species identification.