DOI: 10.5061/dryad.k6djh9w6h
title: Gregarines modulate insect responses to sublethal insecticide residues

description: Throughout their lifetime, insects face multiple environmental challenges
 that influence their performance. Gregarines are prevalent endoparasites
 in most invertebrates that affect the fitness of their hosts, but are
 often overlooked in ecological studies. Next to such biotic factors, a
 current common challenge is anthropogenic pollution with pesticides, which
 causes a major threat to non-target organisms that are readily exposed to
 lethal or sublethal concentrations. In a laboratory study, we investigated
 whether the presence of gregarines modulates the food consumption and
 life-history traits of a (non-target) leaf beetle species, Phaedon
 cochleariae, in response to sublethal insecticide exposure. We show that
 the larval food consumption of the herbivore was neither affected by
 gregarine infection nor sublethal insecticide exposure. Nevertheless,
 infection with gregarines led to a delayed development, while insecticide
 exposure resulted in a lower body mass of adult males and a reduced
 reproduction of females. Individuals exposed to both challenges suffered
 most, as they had the lowest survival probability. This indicates
 detrimental effects on the population dynamics of non-target insects
 infected with naturally occurring gregarines that face additional stress
 from agrochemical pollution. Moreover, we found that the infection load
 with gregarines was higher in individuals exposed to sublethal insecticide
 concentrations compared to unexposed individuals. To counteract the global
 decline of insects, the potential of natural parasite infections in
 modulating insect responses to anthropogenic and non-anthropogenic
 environmental factors should be considered in ecological risk assessment.
description: <b>Study organism and rearing</b>
 A rearing of <i>P. cochleariae</i> was maintained for
 several generations at Bielefeld University under constant climatic
 conditions (20°C, 16 h: 8 h light:dark, 70% r.h.). Adult beetles were
 mixed randomly for mating and groups of 100-200 individuals kept in boxes
 (20 × 20 × 6.5 cm) covered with gauze lids. Every year the rearing
 population was replenished with individuals collected in the field
 (51°51′21″ N, 8°41′37″ E). Larvae and beetles were supplied with leaves of
 non-flowering 8-10-week old cabbage plants (<i>Brassica
 rapa</i> L. spp. <i>pekinensis</i>), which were
 cultivated in pots in a greenhouse (20˚C, 16 h: 8 h light:dark, 70% r.h.).
 For the experiment, only middle-aged leaves were offered, and larvae were
 provided with greenhouse-grown cabbage, while adults received cabbage
 bought from an organic store due to plant shortage.
 <b>General experimental set-up and measurements of larval
 food consumption and life-history traits</b> A
 full-factorial design was set up to test the influences of gregarine
 infection and sublethal insecticide exposure on larval consumption and
 life-history of <i>P. cochleariae</i>. Three-week-old adults
 (about 200 individuals) were provided with cabbage leaves and their
 offspring was subsequently used for the experiment. Female beetles bite
 little cavities in the leaf surface, lay individual eggs in these cavities
 and cover the eggs with secretion. Infection of larvae of <i>P.
 cochleariae</i> with gregarines occurs via the uptake of spores
 excreted by infected conspecifics. Faecal remains can also cover the eggs.
 Thus, after 24 h of oviposition time, eggs were carefully removed from the
 leaves and the secretion and potential faeces removed with a paintbrush
 and tap water. Eggs were placed on fresh cabbage leaves and randomly
 distributed over two rearing boxes, one assigned to the uninfected
 gregarine treatment (G-: <i>N </i>= 325), the other to the
 gregarine infection treatment (G+: <i>N</i> = 320). Hatching
 larvae (G-: <i>N</i> = 195, G+: <i>N</i> = 204)
 were supplied with the respective food sources for four days as described
 below (experimental infection of <i>P. cochleariae</i> larvae
 with gregarines) to ensure a G- or G+ treatment. At the
 fourth day after hatching, the larvae were divided into groups of 5-10
 larvae in large Petri dishes (9 cm diameter) lined with filter paper and
 provided with cabbage leaf pieces (3 × 4 cm). From day five on, half of
 both G- and G+ larvae were assigned to one of the two insecticide
 treatment groups, either receiving no insecticide (I-) or receiving
 λ-cyhalothrin-treated leaf discs (I+) for 48 h (procedure see below),
 resulting in four treatment groups (G-I-: <i>N</i> = 50, G-I+:
 <i>N</i> = 113, G+I-: <i>N</i> = 60, G+I+:
 <i>N</i> = 130). Much higher numbers of I+ animals were set up
 to account for a lower survival of insecticide-treated individuals. From
 day seven after hatching, all individuals were provided with untreated
 cabbage leaves. To investigate the effects of the
 different treatments on consumption, the amount of leaf mass consumed
 within 24 h by each larva was measured. At day 9 after hatching, two
 larvae were randomly selected from each Petri dish. Each larva was weighed
 (micro balance, ME36S, Sartorius AG, Göttingen, Germany), placed in a
 small Petri dish (5.5 cm diameter) lined with moistened filter paper and
 offered a leaf disc (24 mm diameter) of known mass (balance, LA120S-OCE,
 Sartorius AG, Göttingen, Germany). After 24 hours, the larvae were weighed
 again and the remaining leaf discs were scanned (Samsung ProXpress
 SL-M3375FD, Schwalbach/Taunus, Germany; resolution: 600 dpi). The
 remaining leaf area was determined with ImageJ (v 1.52a) and used to
 calculate the consumed leaf mass [initial leaf mass x (initial leaf disc
 area – remaining leaf area)/initial leaf disc area]. Afterwards, these
 larvae remained in their individual Petri dishes and were used for
 counting of gregarines (see below). To test the effects
 of the different treatments on life-history traits, pupae were
 individually placed in small Petri dishes lined with filter paper and the
 time from larval hatching until adult eclosion was noted. The body mass of
 adult individuals was measured 24 hours after adult eclosion and the sex
 of the individuals determined. Adults were kept individually in small
 Petri dishes and supplied with cabbage leaves. Eight days after adult
 eclosion females were mated with males of the same treatment group,
 placing each male to one female for 24 hours. After mating, the pairs were
 separated again, and the number of eggs laid within the subsequent four
 days per female was counted. The hatching rate was determined by counting
 the number of hatched larvae relative to the number of eggs laid.
 Moreover, the survival of all individuals kept under the four treatment
 regimes was noted until day ten after adult eclosion. The experiment was
 ended when adults were 15 days old, while adults can live up to three
 months (Bogdanov-Katjkov 1923). <b>Experimental
 infection of <i>P. cochleariae</i> larvae with
 gregarines</b> To ensure larval infection with
 gregarines (G+ treatment), leaves covered with faeces were taken that had
 been placed in the boxes of the insect rearing stock for 24 hours. Random
 examinations of the rearing stock showed that all tested beetles were
 infected with gregarines and that infectious spores were present in their
 faeces. Larvae of the G- treatment were offered leaves that had been kept
 for 24 hours in a box without conspecifics and were damaged by regular
 cuttings to imitate feeding and thus provide leaves of comparable quality
 to those provided to the G+ group. Such leaves were offered to hatching
 larvae of the respective treatment groups and replaced every 2 days until
 larvae were 4 days old. Microscopy of larvae and adults (see below)
 confirmed that all dissected individuals of the G- treatment
 (<i>N</i> = 40 larvae, <i>N</i> = 46 adults) were
 not infected, while all individuals of the G+ treatment were successfully
 infected with gregarines (<i>N </i>= 40 larvae,
 <i>N</i> = 73 adults). <b>Preparation
 of sublethal λ-cyhalothrin concentration and insecticide exposure
 treatment</b> The pyrethroid
 λ-cyhalothrin was chosen as contact insecticide because it is widely used
 in agriculture, including fields with crops of Brassicaceae. A sublethal
 concentration was prepared from the insecticide LAMBDA WG, which contains
 5% of the active toxin λ-cyhalothrin. The insecticide was dissolved in
 methanol (HPLC-grade, VWR International GmbH, Darmstadt, Germany),
 centrifuged and the supernatant used to prepare a 0.6 mg/L λ-cyhalothrin
 stock solution. This concentration was considered as sublethal, as less
 than 50% of the larvae died during the exposure period (following the
 definition by de França et al. 2017). In an agricultural system, this
 concentration is approximately less than half of the application
 recommended by the supplier and may thus occur in areas close to treated
 fields as residue. The experimental larvae were fed on day five and six
 after hatching with cabbage leaf discs (24 mm diameter) treated with 76.8
 µl methanol (I-) or λ-cyhalothrin solution (I+). The surfaces of the leaf
 discs were evenly covered with the respective solutions using a pipette
 and leaf discs were kept under a fume hood until evaporation of the
 solvent (at least 20 min) before offering them to the larvae. Depending on
 the number of larvae, two to four leaf discs were offered per Petri dish
 to provide food <i>ad libitum</i> and after one day the leaf
 discs were exchanged by discs of the respective treatments for another
 day. <b>Counting of
 gregarines</b> Subgroups of both larvae and
 adults of <i>P. cochleariae</i> were examined to test whether
 the number of gregarines depends on the different treatments and
 developmental stage of the host. At day 12 after larval hatching, a subset
 of larvae of the different treatments (two larvae per Petri dish, used for
 consumption assay, <i>N</i> = 20 larvae per treatment group)
 was taken and individuals were frozen (-20 °C). Likewise, adult males and
 females of each treatment group (G-I-: <i>N</i> = 9 females,
 <i>N</i> = 12 males; G-I+: <i>N</i> = 12 females,
 <i>N</i> = 13 males; G+I-: <i>N</i> = 12 females,
 <i>N</i> = 15 males; G+I+: <i>N</i> = 26 females,
 N = 20 males) were randomly selected and frozen at day 15 after adult
 eclosion. Afterwards, individuals were thawed, the midgut dissected,
 spread on a microscope slide in 25 µl sodium phosphate buffer (0.1 M, pH =
 7.2), and the number of gregarines (likely as trophozoites and gamonts)
 counted under a light microscope at 200 to 400 times magnification (ZEISS
 Axiophot, Carl Zeiss Microscopy GmbH, Jena, Germany) (ESM Fig.
 S2). <b>Statistical analyses</b>
 All statistical analyses were performed in R version 3.6.3 (R
 Core Team 2020) with RStudio version 1.2.5033 (RStudio Team 2019). Data
 were analysed using linear models (lm), generalised linear models (glm)
 (package MASS; Venables and Ripley 2002) and cox proportional hazards
 model (package survival; Therneau 2020). Model assumptions for lms and
 glms (normal distribution and homoscedasticity of residuals) and cox
 proportional hazard models (proportional hazard and influential
 observations) were checked using diagnostic plots. Stepwise backward model
 selection was applied to obtain the minimal adequate models. Based on F
 test or Chi-square test results (package MASS; Venables and Ripley 2002),
 non-significant (<i>P</i>-value &gt; 0.05) interaction
 terms and/or predictors were excluded from the models.
 The effects of the predictors gregarine treatment and insecticide
 treatment as well as their interaction on development time (from larval
 hatching to adult eclosion) were tested using a glm (poisson distribution,
 identity link function). The influences of the predictors larval body
 mass, gregarine treatment and insecticide treatment and their interaction
 on the food consumption of larvae were analysed using a lm. The effects of
 the predictors gregarine treatment, insecticide treatment as well as their
 interaction on the adult body mass were analysed separately for males and
 females using lm. Glms were performed to investigate the effects of
 gregarine treatment, insecticide treatment and their interaction on the
 number of eggs laid by adult females (poisson distribution, log link
 function) and the hatching rate of larvae [binomial family
 (success/failure of hatching), logit link function]. To investigate the
 effects of insecticide treatment on the gregarine load in the larval stage
 a glm (poisson distribution, identity link function) was calculated. For
 the gregarine load in adults a glm (poisson distribution, identity link
 function) with insecticide treatment and sex as predictors was performed.
 The influences of the predictors gregarine treatment, insecticide
 treatment and their interaction on survival probability were tested using
 a cox model, followed by pairwise log rank post hoc tests. Survival data
 were plotted using Kaplan-Meyer curves (package survival; Therneau
 2020). R Core Team (2020) R: a language and environment
 for statistical computing. R Foundation for statistical computing, Vienna,
 Austria. https://www.R-project.org/. R Studio Team (2019) RStudio: integrated development for R. RStudio, Inc., Boston, MA. http://www.rstudio.com/. Therneau T (2020) A package for survival analysis in R. R package version 3.2-7. https://CRAN.R-project.org/package=survival. Venables WN, Ripley BD (2002) Modern applied statistics with S, 4. edn. Springer, New York.