DOI: 10.5061/dryad.gmsbcc2js
title: Philornis parasitism: impact on nestlings and risk factors involved

description: Parasitic botfly larvae (Philornis ssp., Diptera: Muscidae) are found in
 nests of several bird taxa, although prevalence and nestling tolerance
 vary considerably among species. Here we describe patterns of botfly
 infestation in blue-black grassquit (Volatinia jacarina) nestlings. We
 identified the most typically affected nestling body parts and assessed
 parasite prevalence, impact on nestling survival, changes in nestling body
 shape and mass index. Additionally, we tested whether climatic conditions,
 nest morphology and habitat characteristics are associated with larvae
 abundance. Blue-black grassquits had low breeding success (15%), but most
 failures resulted from predation by vertebrate predators. We estimated
 that only 1% of nestlings died due to botfly infestation, and the number
 of larvae in nestling body did not affect nest success. Infected chicks
 exhibited a higher body mass to tarsus length ratio, and higher tarsus
 asymmetry. Previous studies indicate that adult grassquits with a higher
 body mass index have lower dominance status and mating success. Thus, we
 argue that although botflies had a small impact on offspring survival,
 they may reduce fitness in adulthood. There was no evidence that
 environmental conditions and nest morphology are linked to the number of
 larvae on nestlings. Territories with higher food supply had lower
 infestation rates. Possibly, food-rich habitats allow parents to invest
 more time in offspring care (brooding nestlings), thus protecting them
 from fly attacks. The present study brings to light new perspectives
 concerning bird-botfly interaction
description: <strong>Study site and species</strong>
 This study was carried out within the University of Brasília
 campus, in central Brazil (15°45'S; 47°52'W), in an area of 20
 ha. The vegetation is classified as Cerrado <i>sensu
 stricto</i> (tropical savanna) with high plant diversity (Assunção
 and Felfili 2004, Aguilar et al. 2008). We monitored breeding activities
 of blue-black grassquits between November to April, across two breeding
 seasons (2017-2018; 2018-2019). Throughout the breeding season, males
 blue-black grassquits defend territories and attract mates by performing
 multimodal displays (Manica et al. 2016, Manica et al. 2017). Both sexes
 build the nest and provide parental care. The small cup-shaped nests are
 placed in the forks of shrubs (more frequently) or in dense grass
 undergrowth (Carvalho et al. 2007, Aguilar et al. 2008). Predation is
 considered the main factor leading to lack of breeding success (Almeida
 and Macedo 2001, Carvalho et al. 2007, Aguilar et al. 2008, Dias et al.
 2010). Cheating behaviors, such as extra pair paternity and intraspecific
 brood parasitism, occur in blue-black grassquits (Carvalho et al. 2006,
 Manica et al. 2016). Little is known about botfly infection in this
 species, but literature reports indicate that they are infected by the
 subcutaneous<i> P. glaucinis</i> and <i>P. trinitensis
 </i>(Teixeira 1999). <strong>Data
 collection</strong> We searched for nests by
 slowly walking across the field site at least twice each week, inspecting
 the vegetation and watching for birds carrying nesting materials. New and
 active nests were checked at intervals of up to three days, until chicks
 fledged or the nest was lost to predation. Incubation and nestling periods
 lasted up to 10 days each (Carvalho et al. 2007).  If eggs were present in
 a nest on a given day and eggs hatched on the following day, we assumed
 that hatching had occurred on the second day. For chicks hatching after a
 checking interval of two-three days, or nests found in the nestling period
 (20 of 180), we estimated hatching day by comparisons with chicks of known
 age. The disappearance of eggs before hatching or of nestlings before
 seven days of age, associated with a damaged nest, was attributed to nest
 predation. Nestling death due to botfly larvae infestation was assumed
 when nestlings were found dead in the nest and larvae were found in the
 nest or nestling. Nest desertion was assumed when parents no longer cared
 for the eggs, which remained in the nest &gt; 10 days. Whenever eggs
 were deserted, we collected and opened them to check for development.
 After this inspection, eggs were classified as “infertile” if no embryo
 was found, or “death in development” if we found a dead embryo. Nest
 success was assumed when nestlings disappeared from the nest at or later
 than 7 days post-hatch and no signs of predation were detected
 (<i>i.e. </i>the nest remained intact in the
 vegetation). Nestling body condition was recorded up to
 three times for each nestling, and included mass (taken with a 10g, 0.1g
 resolution Pesola spring scale), both left and right tarsus length
 (digital caliper Mitutoyo 500-196-30B, 0.01 mm resolution), and number of
 larvae and their location on the nestling´s body (we did not identify
 larvae to the species level). A body mass index was calculated by dividing
 nestling weight by average tarsus length. In adult grassquits, it has been
 found that body mass index correlates negatively with intestinal parasite
 load (Costa and Macedo 2005, Aguilar et al. 2008) and social dominance
 (Santos et al. 2009). Nest body asymmetry was calculated based on the
 absolute difference between left and right tarsus lengths. The location of
 larvae was mapped onto the nestling´s body areas: head-neck, wings, legs,
 and main body (see figure 1); large larvae could occupy more than one
 area. We did not band nestlings at the nest since this could influence
 natural predation rate by increasing the contrast between nestlings and
 nest background material. Climatic data were obtained
 from the open database provided by the Brazilian meteorological institute
 (Instituto Nacional de Meteorologia - INMET), which provided a regional
 sampling location less than 10 km from the study site. For each nest, we
 averaged the daily rainfall and temperature for a period encompassing 14
 days, from seven days before and after hatching. When
 nests were no longer active, they were collected for measurements
 associated with their architecture, after which they were deposited in the
 museum collection Coleção Ornitológica Marcelo Bagno, at Universidade de
 Brasília. We used two variables to characterize nest architecture: nest
 wall density and nest wall openness. Nest wall density was calculated as
 the ratio of nest weight to wall volume (mg/mm<sup>3</sup>). 
 Nest weight was obtained with a high precision balance (Shimadzu BL320H,
 1mg resolution) after the nest was air dried at 75ºC for 24h. Nest wall
 volume was estimated as the difference between external and internal nest
 wall volumes. Volume was estimated using the semi-ellipsoid volume formula
 V = 2/3 × π × a × b × c, where a and b are perpendicular measurements of
 nest outer/inner diameter and c is nest height/depth. Nest wall openness
 was estimated as the average of four measurements of nest wall openness
 taken on different sides of the nest. A detailed description of the method
 is provided in Biagolini-Jr and Macedo (2019). In brief, photos of the
 nest were taken with a white styrofoam ball (50mm in diameter) placed
 inside the nest chamber. Then, with an image editor software (GIMP version
 2.10) we cropped the styrofoam ball image section and converted it to a
 black and white scale. Using the R package bwimage (Biagolini-Jr 2019), we
 estimated nest wall openness as the proportion of white pixels relative to
 total number of image pixels. We assessed food
 availability and vegetation density in five spots at distances of 3 m from
 the nest within two weeks after nestlings fledged. We did not demarcate
 parental territories, but assume that the sampled spots had a high
 probability of falling within territories (Aguilar et al. 2008). We
 estimated the abundance of seed resources by averaging the number of seed
 inflorescences counted in 50 x 50 cm grids placed at each of the five
 spots (Manica et al. 2014). Vegetation density was estimated in five plots
 of 30x100 cm, by adapting the Zehm et al. (2003) method. In summary, a
 photograph was taken of the vegetation against a panel of 100 x100 cm
 white cloth placed perpendicularly to the ground on the largest side of
 the plot. The photograph was converted to a pure black and white image
 (GIMP version 2.10). Vegetation density was estimated as the proportion of
 black pixels relative to total number of pixels (Biagolini-Jr and Macedo
 2019). Host nest density was estimated as the number of
 grassquit nests, within a radius of 50 meters of the focal nest being
 considered, with clutches that hatched in the period of 10 days before and
 after nest hatching date at the focal nest. We chose this range because it
 encompasses both the nestling period and the botfly pupation period from a
 possible previously infected nest (Saravia-Pietropaolo et al. 2018). We
 used the R package geosphere Version 1.5 (Hijmans et al. 2019) to
 calculate distances between nests.