DOI: 10.5061/dryad.sj3tx96c6
title: Different strategies between queens and workers against fungal pathogens in the termite Reticulitermes chinensis

description: Social insects are prone to pathogen infection because of high
 exposure rates from social interactions. However, it remains
 unclear whether queens have enhanced pathogen resistance, since
 reproduction is largely confined to queens. Here, we
 used a natural host-pathogen system, the subterranean termite
 Reticulitermes chinensis and the entomopathogenic fungus
 Metarhizium anisopliae, to investigate the differences in
 allo-grooming, locomotion, and immune gene expression between queens and
 workers against pathogen infection. We found that fungal infection
 significantly reduced survival in both queens and workers. Infected queens
 received significantly more grooming time from sanitary nestmates than
 infected workers, but they returned much less grooming time to sanitary
 nestmates than infected workers. Infection resulted in a reduction in the
 average locomotion speed and distance of queens but had no effect on
 worker locomotion. Infection resulted in upregulated expression of two
 immune genes (termicin and transferrin), two antioxidant genes
 (CAT and SOD), and phosphate genes CYP450 in queens but not
 in workers. Our results indicated that eusocial termites
 evolved strategies that
 prioritize the reproductive castes
 welfare in defending against the pathogen
 infection to ensure continued reproduction and colony
 persistence.
description: <strong>Study termites</strong>:<strong>
 </strong>Two colonies of <em>R.
 chinensis</em><em> </em>were<em> </em>collected from Lion Mountain, Wuhan, China in 2022. Adult termites were split out from the cracks of dead wood and each 50 individuals (48 workers and 2 soldiers) were housed in a 6-cm Petri dish containing moist filter paper as a food and water source. Each colony was divided into 60 sub-colonies (120 sub-colonies in total). All sub-colonies were kept in a climatic chamber (25<sup>o</sup>C, 70% relative humidity, 12 h day/night cycle). Once a week, we checked the consumption of a piece of moist filter paper and provided corresponding supplements. Previous studies have shown that workers of <em>R. chinensis</em> are able to develop into neotenics (supplementary reproductive) when the primary queen dies or is lost (Su et al., 2015). Here, in our observations, after about three weeks, the workers will differentiated into neotenics. We classed the neotenics as queens for all experiments. These all sub-colonies with a queen were used for the following experiments. <strong>Fungal pathogens</strong>:<strong> </strong>We used the entomopathogenic fungus <em>M. anisopliae</em> (strain NRRL13969) to infect the focal workers and queens. The fungus was cultivated on potato dextrose agar (Solarbio, China) for 3 weeks, and used 0.1% Tween 80 to collect the conidial suspensions, then stored at 4°C. All conidial suspensions had a germination rate of &gt;95% as determined directly before starting each experiment. We exposed individual termite workers by applying 0.35μl droplet of a suspension of 10<sup>4</sup> conidia/ml in 0.1% Tween 80 (infected treatment). and the control treatment consisted of an application of a 0.35μl droplet of a 0.1% Tween 80 only. Subsequently, all termites were refrigerated at 4°C for one hour to slow their movements and to precipitate the conidia onto their cuticle before starting the experiments (Traniello et al. 2002). <strong>Survival</strong>:<strong> </strong>To determine the effect of infection with <em>M. anisopliae</em> on the different termite castes, 100 workers and 100 queens were randomly picked out from 100 sub-colonies (50 sub-colonies per colony), and each termite individual was assigned to one of four treatments: control worker (n=50), control queen (n=50), infected worker (n=50), and infected queen (n=50). All of these treatments were reared individually in 3.5-cm Petri dish with moist filter paper for providing food and water, and kept in a constant climatic chamber (25°C, 70% relative humidity, 12 h day/night cycle). The number of dead termites was recorded daily over 12 days, after which the dead termites were removed.<strong> </strong> <strong>Locomotion and allogrooming</strong>:<strong> </strong>To observe the locomotion and allogrooming behavior of workers and queens, we randomly picked 8 individuals (7 workers and 1 queen) from each sub-colony to be placed into a new 3.5-cm Petri dishes, totally set 12 groups (6 control groups and 6 infected groups). Of these 8 individuals, 6 workers were provided to act as caregivers, a single queen and another worker were infected with Tween 80 or <em>M. anisopliae</em>. Each individual was marked by a unique paint color dot on its thorax (Street Graffiti, purchased by MINISO, Guangzhou China). All replicates were kept in a constant climatic chamber (25<sup>o</sup>C, 70% relative humidity, 12 h day/night cycle) for three days to allow them to adapt to their new environment. Every 24h, we checked the color code of each individual, and if the paint of an individual was missing, we re-marked it immediately. After the three-day adaptation period in the new Petri dish, one queen and one worker from each of 6 groups were treated with conidia-free 0.1% Tween 80, and one queen and one worker from each of the other 6 groups were infected with <em>M. anisopliae </em>as described above. The behaviors of these 12 groups were recorded with a camera (AX100E, SONY) for 30 min. The mortality rates of control workers, control queens, infected workers, and infected queens of these 12 groups were recorded after the behavioral measurements were taken. Ultimately, the video recordings were used to analyse the behaviors of allogrooming and locomotion related data with the help of a computerized EthoVision XT 15 tracking system (Noldus Information Technology, Beijing China): 1) grooming-received: the total duration time of control and infected individual received grooming from other 7 nestmates; 2) grooming-given: the total grooming time given to the other 7 nestmates by the control and infected individual; 3) velocity: the average moving speed of control and infected individuals in the petri dish during 30-min observation; 4) distance: the total distance that the focal individual covered during 30-min observation. EthoVision was also used to generate motion trajectories and heatmaps for the distribution of the termites around the test arenas during each of the 30-min recordings (for details see Appendix S1). <strong>Quantitative real-time PCR (RT-qPCR)</strong>:<strong> </strong>The expression of six genes <em>termicin</em>, <em>transferrin</em>,<em> </em><em>cytochromeP450</em> (<em>CYP450</em>), <em>glutathione S-transferase</em> (<em>GST)</em>, <em>superoxide dismutase</em> (<em>SOD</em>), and <em>catalase</em><em> </em>(<em>CAT</em>) were analyzed in control workers, control queens, infected workers, and infected queens by real-time PCR. We randomly picked out 40 workers and 8 queens from 8 sub-colonies, of which 20 workers and 4 queens were treated with conidia-free 0.1% Tween 80, and another 20 workers and 4 queens were infected with <em>M. anisopliae</em><em>. </em>Four replicates (each worker replicate containing a pool of five workers and queen replicate containing one queen) were set up for each treatment (2 replicates per colony). The gene expressions were measured 48h after initial fungal infection. Each sample was crushed in a 1.5-mL centrifuge tube with liquid nitrogen, using sterilized disposable tissue grinding pestles. Total RNA was extracted from the whole body of workers and queens using RNeasy mini kit (QIAGEN), respectively. After RNA extraction, the quality and quantity (purity of protein and salt) were analysed using a NanoDrop Lite spectrophotometer (Model: ND Lite-4470). Then the cDNA was synthesized from the RNA by GoScriptTM Reverse Transcription Mix, Oligo(dT) (Promega), and was preserved at -20˚C. The quantitative real-time PCR (qRT-PCR) was performed on the Applied Biosystems® StepOne™ system (Thermo) with QuantiNovaTM SYBR® Green PCR Kit (Qiagen). All the procedures were implemented in accordance to the manufacturer’s protocol. Since <em>β-actin </em>is considered the most reliable reference gene for <em>Reticulitermes</em> termites (Tong et al. 2009; Ishitani and Maekawa, 2010), this was included in our experiment. Relative gene expression levels were calculated using a typical 2<sup>-ΔΔCt</sup> method. We performed four biological replicates and four experimental replicates for all RT-qPCR experiments. The primers used for real-time PCR are listed in Table 1. <strong>Statistical analysis</strong>:<strong> </strong>All statistical analyses were performed with IBM SPSS Statistics version 22.0 and the program R v3.6.3. All graphics were produced with GraphPad Prism 8.0. Cox proportional regression was used to evaluate the effect of variables (separately reared and group-reared) on the survival of termites. Since the survival curves of workers and queens in different treatments (control vs. infected) were crossing, a two-stage method (Qiu & Sheng, 2008) was used using R’s TSHRC package. For the allogrooming behavior, locomotion behavior, and immune-gene expression, we applied the generalized linear mixed model (GLMM) with a normal distribution to analyze the effects of castes (workers vs. queens) and treatment types (control vs. infected), as well as their interaction, respectively. In these three models, the castes and treatment types as the fixed factors, and the colony as the random factor. In addition, post hoc pairwise comparisons with LSD tests were performed to examine the differences of allogrooming, locomotion, and immune gene expression in workers and queens under different treatments (control and infected).
description: # \*Different strategies between queens and workers against fungal
 pathogens in the termite *Reticulitermes chinensis*   **Datasets
 included:** a. **Survival** b. **Allogrooming and Locomotion** c. **Gene
 expression** \** ** **DATA-SPECIFIC INFORMATION FOR: Survival** 1\. Number
 of variables: 6 2\. Number of cases/rows: 24 3\. Variable List: *
 Individual_Group: individual rearing or group rearing. *
 Days: day of the study. * num_infected_queens: number of
 infected queens death per day * num_infected_workers: number
 of infected workers death per day * num_control_queens:
 number of control queens death per day *
 num_control_workers: number of control workers death per day 4\.
 Specialized formats or other abbreviations used: None   **DATA-SPECIFIC
 INFORMATION FOR: Allogrooming and Locomotion** 1\. Number of variables: 11
 2\. Number of cases/rows: 24 3\. Variable List: * Control_Infected: the
 termite infected with Tween 80 (control) or *M. anisopliae* (infected).
 * Workers_Queens: workers or queens. *
 Repetitions: the number of biological repetition for termites.
 * Grooming-received from caregivers (s): the grooming time
 of focal worker or queen received from six caregivers, in seconds.
 * Grooming-given to caregivers (s): the grooming time of
 focal worker or queen  given to six caregivers, in seconds. 
 * Grooming-given to focal queen (s): the grooming time of focal worker
 given to focal queen, in seconds. * Grooming-given to focal
 worker (s): the grooming time of focal queen given to focal worker, in
 seconds. * Total duration of grooming-received (s): the
 total grooming time of focal worker (queen) received from six caregivers
 and focal queen (worker), in seconds. * Total duration
 Grooming-given (s): the total grooming time of focal worker (queen) given
 to six caregivers and focal queen (worker), in seconds. *
 Velocity (cm/s): average velocity in the petri dish during 30-min
 observation, in cm/s * Distance (cm): total distance in the
 petri dish during 30-min observation, in cm 4\. No data: / 5\. Specialized
 formats or other abbreviations used: None   **DATA-SPECIFIC INFORMATION
 FOR: Gene expression** 1\. Number of variables: 5 2\. Number of
 cases/rows: 48 3\. Variable List: * Gene name: the name of genes
 * Control_Infected: the termite infected with Tween
 80 (control) or *M. anisopliae* (infected). * Repetitions:
 the number of biological repetition for termites. * Workers:
 the gene expression of workers. * Queens: the gene
 expression of queens. 4\. Specialized formats or other abbreviations used:
 None