DOI: 10.5061/dryad.pvmcvdnp8
title: Starvation increases susceptibility to bacterial infection and promotes systemic pathogen proliferation in Drosophila melanogaster females

description: Defense against pathogens and parasites requires substantial investment of
 energy and resources on part of the host. This makes the host immune
 function dependent on availability and accessibility of resources. A
 resource deprived host is therefore expected to be more susceptible to
 infections, although empirical results do not always align with this
 prediction. Limiting host access to resources can additionally impact
 within-host pathogen numbers, either directly by altering the amount of
 resources available to the pathogens for proliferation or indirectly by
 altering the efficiency of the host immune system. We tested for the
 effects of host starvation (complete deprivation of resources) on
 susceptibility to bacterial pathogens, and within-host pathogen
 proliferation, in Drosophila melanogaster females. Our results show that
 starvation increases post-infection mortality of the host, but in a
 pathogen-specific manner. This increase in mortality is always accompanied
 by increased within-host pathogen proliferation. We therefore propose that
 starvation compromises host resistance to bacterial infections in
 Drosophila melanogaster females thereby increasing susceptibility to
 infections.
description: <strong>1. Fly populations and general
 handling</strong> Experiments reported here were
 carried out on flies from a large, outbred laboratory adapted population
 of <em>Drosophila melanogaster</em>, LH (Chippindale and Rice
 2001, Prasad et al., 2007, Nandy et al., 2012). The LH population is
 maintained on a 14-day discrete generation cycle, at 25
 <sup>O</sup>C temperature and 12:12 hour light-dark cycle, on
 cornmeal-molasses-yeast medium, at a census size of about 1900 adults. The
 flies are maintained in vials (95 mm height and 25 mm diameter); each
 generation starts with setting up of 60 vials with 150 eggs each on 8-10
 ml of food medium. 12 days post-egg laying (PEL), by which time most
 adults have eclosed, adults from different vials are mixed together and
 redistributed into 60 vials with 16 females and 16 males in each vial. The
 vials are supplied with limiting live dietary yeast supplement, and on
 14<sup>th</sup> day PEL, the adults are transferred to fresh
 vials and allowed to oviposit for 18 hours to start the next generation.
 Vials with eggs greater than 150 undergo egg-culling to maintain the
 specified egg density in vials and avoid crowding.
 <strong>2. Derivation of experimental
 flies</strong> 12-day PEL adults were transferred
 to plexiglass cages (14 cm length × 16 cm width × 13 cm height) at a
 density of 1000-1200 flies, and the cages are provided with standard food
 medium in 60 mm Petri plates. For collection of eggs for setting up
 experiments, cages are provided with a fresh food plate, supplemented with
 <em>ad libitum</em> live yeast supplement, for 48 hours. This
 is done to encourage egg production and laying in the females. Following
 this, a fresh food plate is provided to the cages and 12-14 hours later
 eggs are collected off these plates (using moist paint brushes on 1.5%
 agar gel) and seeded into food vials (with 8-10 ml of food medium) at an
 exact density of 150 eggs per vial. The number of vials set up in this
 manner depends upon the requirement for a particular experiment. These
 vials are then incubated under standard conditions (detailed above) for
 egg to mature into larvae and then into adults. On
 10<sup>th</sup> day PEL, during the eclosion peak, adults are
 collected in unmated state within 5-6 hours of eclosion, and housed in
 single-sex vials (each with 1-2 ml of food medium) at constant density of
 8 females per vial or 10 males per vial. Flies are housed in these vials
 till further manipulation/experimentation.
 <strong>3. Bacterial handling and infection
 protocol</strong> The bacterial isolates are
 preserved as glycerol stocks at -80 <sup>O</sup>C. To obtain
 live bacterial cells for infections, 10 ml lysogeny broth (Luria Bertani
 Broth, Miler, HiMedia) is inoculated with glycerol stocks of the necessary
 bacterium, and incubated overnight with aeration (150 rpm shaker
 incubator) at suitable temperature. 100 microliters from this primary
 culture is inoculated into 10 ml fresh lysogeny broth and incubated for
 the necessary amount of time to obtain confluent
 (OD<sub>600</sub> = 1.0-1.2) cultures. The bacterial cells are
 pelleted down using centrifugation and resuspended in sterile
 MgSO<sub>4</sub> (10 mM) buffer at optical density
 (OD<sub>600</sub>) of 1.0. Flies are infected, under light
 CO<sub>2</sub> anaesthesia, by pricking them on the
 dorsolateral side of their thorax with a 0.1 mm Minutien pin (Fine
 Scientific Tools, USA) dipped in the bacterial suspension. Sham-infections
 (injury controls) are carried out in the same fashion, except by dipping
 the pins in sterile MgSO<sub>4</sub> (10 mM) buffer. Across
 three experiments reported in this paper, five pathogens in total were
 used in this study, namely, (a)  
 <em>Enterococcus faecalis</em> (Lazzaro et al., 2006),
 incubation temperature 37 <sup>O</sup>C;
 (b)   <em>Erwinia carotovora carotovora</em>, strain
 Ecc15 (Martins et al., 2013), incubation temperature 29
 <sup>O</sup>C; (c)   <em>Providencia
 rettgeri</em> (Short and Lazzaro 2010), incubation temperature 37
 <sup>O</sup>C; (d)   <em>Pseudomonas
 entomophila</em>, strain L48 (Vodovar et al., 2005, Mullet et al.,
 2012), incubation temperature 27 <sup>O</sup>C; and,
 (e)   <em>Staphylococcus succinus</em>, strain PK-1
 (Singh et al., 2016), incubation temperature 37
 <sup>O</sup>C. These pathogens are reported
 natural pathogens of <em>D. melanogaster</em> and previous
 studies have demonstrated that these cause lethal infection in flies in
 the laboratory following systemic infection (Dionne and Schneider 2008,
 Troha and Buchon 2019). Some of the strains used here were isolated from
 wild-caught flies. Additionally, these pathogens all have intermediate
 virulence: they are neither completely benign nor completely lethal to the
 host. This is important since excessive lethality due to infection, or a
 lack thereof, can mask the subtle effects of starvation on post-infection
 survival. Furthermore, two of the pathogens used – <em>E.
 faecalis</em> and <em>S. succinus</em> – are
 Gram-positive bacteria, while the rest – <em>P.
 entomophila</em>, <em>E. c. carotovora</em>, and
 <em>P. rettgeri</em> – are Gram-negative bacteria. Since
 bacteria of different Gram-character elicit different immune responses
 from the insect hosts, with some overlap and crosstalk (Lemaitre and
 Hoffman 2007, Dionne and Schneider 2008, Vallet-Gely et al., 2008, Buchon
 et al., 2014), we included bacterial pathogens of both Gram-character in
 our experiments. Previous studies have shown that in
 <em>D. melanogaster</em>, defense against <em>E.
 faecalis</em> remains unaffected by brief period of starvation prior
 to infection (Brown et al., 2009) and dietary restriction (Ayres and
 Schneider 2009), while brief starvation before infection improves survival
 and reduces systemic pathogen loads for <em>E. c.
 carotovora</em> (Brown et al., 2009). Additionally, low-protein
 diets increase susceptibility of flies to <em>P.
 entomophila</em> (Kutzer et al., 2018) and high-carbohydrate diets
 increase systemic pathogen loads following infection with <em>P.
 rettgeri</em> (Unckless et al., 2015).
 <strong>4. Systemic bacterial load
 estimation</strong> To measure the systemic
 bacterial load, females are first surface sterilized using 70% ethanol for
 1 minute and 30 seconds, twice. Females are then washed in sterile
 distilled water for 30 seconds and dried using autoclaved tissue paper.
 Females are then transferred individually to 1.5 ml vials (microcentrifuge
 tubes) containing 50 or 75 microliters (depending upon pathogen used for
 infection) of sterile MgSO<sub>4 </sub>(10 mM) buffer. Females
 are homogenized individually in these vials using a motorized pestle for
 50-60 seconds. This homogenate is serially diluted (1:10 dilutions) 8
 times in sterile MgSO<sub>4</sub> (10 mM) buffer. 10
 microliters from each dilution, and the original homogenate, are spotted
 onto a lysogeny agar plate (2% agar, Luria Bertani Broth, Miler, HiMedia).
 The plates are incubated at required temperature for 8-12 hours (depending
 upon pathogen used for infection), and the number of colony forming units
 (CFUs) in each dilution is counted. The number of CFUs in the countable
 dilution (30 ≤ CFUs ≥ 300) is multiplied by appropriate dilution factor to
 obtain the bacterial load for each individual female.
 <strong>5. Experiment 1: Effect of starvation and sexual
 activity on post-infection survival of females</strong>
 Unmated females and males were obtained following the protocol
 described above. On day 12 PEL, half of the females were randomly assigned
 to ‘unmated’ treatment and the rest to ‘mated’ treatment. Females in the
 ‘mated’ treatment were combined with males in fresh food vials (1-2 ml
 standard food medium) in groups of 8 females and 10 males per vial, and
 allowed to mate for 4 hours (it was visually confirmed that each female
 had mated at least once). Following this, the females were lightly
 anesthetized and infected with bacterial pathogens (or sham-infected)
 following the infection protocol described above; males were discarded.
 Females from the ‘unmated’ treatment were similarly infected (or
 sham-infected). Following infections, half of the females from both these
 treatments were housed in vials with 1-2 ml of standard food medium (‘fed’
 treatment), and the remaining were housed in vials with 1-2 ml 2%
 non-nutritive agar gel (‘starved’ treatment). This produced four
 experimental treatments: (a)   <strong>Unmated,
 Fed (UF):</strong> 10 vials of infected and 5 vials of sham-infected
 females, each vial with 8 females; (b)  
 <strong>Unmated, Starved (US):</strong> 10 vials of infected
 and 5 vials of sham-infected females, each vial with 8 females;
 (c)   <strong>Mated, Fed (MF):</strong> 10 vials of
 infected and 5 vials of sham-infected females, each vial with 8 females;
 and, (d)   <strong>Mated, Starved
 (MS):</strong> 10 vials of infected and 5 vials of sham-infected
 females, each vial with 8 females. Note that in this
 experiment females were subjected to starvation from the time of
 infection. The vials were monitored for mortality every 4-6 hours, for 96
 hours post-infection (HPI); alive flies were shifted to fresh food/agar
 vials at 48 HPI. This experiment was carried out for five bacterial
 pathogens – <em>E. faecalis</em>, <em>E. c.
 carotovora</em>, <em>P. rettgeri</em>, <em>P.
 entomophila</em>, and <em>S. succinus</em> – and
 replicated thrice for each pathogen. In each replicate, 320 females were
 subjected to infection (80 females x 4 treatments) and 160 females were
 subjected to sham-infection (80 females x 4 treatments). Uninfected
 controls (no infection, no sham-infection) were not included in the
 experimental design since preliminary experiments showed that uninfected
 females and sham-infected females do not differ in terms of
 survival. <strong>6. Experiment 2: Effect of
 starvation and sexual activity on systemic bacterial load in infected
 females</strong> Following a protocol identical
 to that of Experiment 1, females were distributed into four treatments
 (UF, US, MF, and MS, as described above) and infected; 100 infected
 females in each treatment and 30 sham-infected females in each treatment.
 Following infections, females were housed in plexiglass cages (14 cm
 length × 16 cm width × 13 cm height), with all females from a particular
 treatment in a single cage; infected and sham-infected females were housed
 in separate cages. (Cages of ‘fed’ treatments were supplied with standard
 food medium in 60 mm Petri plates and cages of ‘starved’ treatments were
 supplied with 2% non-nutritive agar gel in 60 mm Petri plates.) At 4 and
 10 HPI, 12 <em>infected</em> females were randomly aspirated
 out of cages for each treatment and the systemic bacterial load was
 measured for individual females following the CFU enumeration protocol
 described above. Additionally, at 10 HPI, 6 <em>sham-infected
 </em>females were randomly aspirated out of cages for each treatment
 and the systemic bacterial load was measured for individual females. This
 experiment was carried out for three bacterial pathogens – <em>E.
 faecalis</em>, <em>P. rettgeri</em>, and <em>P.
 entomophila</em> – and replicated thrice for each pathogen. In each
 replicate, systemic bacterial load was measured for 96 individual
 <em>infected</em> females (12 females × 2 time-points × 4
 treatments) and 24 individual <em>sham-infected</em> females
 (6 females × 1 time-point × 4 treatments). Sham-infected females from any
 treatment did not yield any CFUs during any of the experimental runs (3
 pathogens × 3 replicates).
description: # Starvation increases susceptibility to bacterial infection and promotes
 systemic pathogen proliferation in Drosophila melanogaster females
 [https://doi.org/10.5061/dryad.pvmcvdnp8](https://doi.org/10.5061/dryad.pvmcvdnp8) ## Description of the data and file structure The file ("survival_data.xlsx") was generated in 2018-2019 by Aabeer Basu and others at the Evolutionary Biology Lab, Department of Biological Sciences, IISER Mohali. The file ("bacterial_load_data.xlsx") was generated in 2021 by Aabeer Basu and others at the Evolutionary Biology Lab, Department of Biological Sciences, IISER Mohali. ### Files and variables #### File: README\_bacterialload.txt **Description:** 1. Title of Dataset: "Effect of resource limitation on host post-infection survival and systemic pathogenic growth" 2\. Author Information A. Principal Investigator Contact Information Name: Prof. N. G. Prasad Institution: Indian Institute of Science Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India. Email: [prasad@iisermohali.ac.in](mailto:prasad@iisermohali.ac.in) B. Associate or Co-investigator Contact Information Name: Aabeer Basu Institution: Indian Institute of Science Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India. Email: [aabeerkbasu@gmail.com](mailto:aabeerkbasu@gmail.com) 3\. Duration of data collection: July - August 2021 4\. Geographic location of data collection: Mohali, Punjab, India 5\. Information about funding sources that supported the collection of the data: IISER Mohali, Govt. of India. ##### Variables * DATA & FILE OVERVIEW File Name:"bacterial_load_data.xlsx" Note: This file contains five separate tabs, each containing data from from experiments using a individual pathogen: Tab 1. "E.faecalis" Tab 2. "P.entomophila" Tab 3. "P.rettgeri" Tab 1. "E.faecalis" 1\. Number of variables: 7 2\. Number of cases/rows: 289 3\. Variable List: Pathogen (Pathogen used to infect the hosts; "Ef" denotes E. faecalis) Replicate (Replicate identity; "A", "B", "C") Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) HPI (Time in hours at which systemic bacterial load was measured; "4" denotes 4 hours post-infection, "10" denotes 10 hours post-infection) FlyID (Identity number of individual fly for which systemic bacterial load was measured) Log2CFU (Log (base 2) transformed value of systemic pathogen load (CFU*Dilution factor) estimated for each individual fly) Tab 2. "P.entomophila" 1\. Number of variables: 7 2\. Number of cases/rows: 289 3\. Variable List: Pathogen (Pathogen used to infect the hosts; "Pe" denotes P. entomophila) Replicate (Replicate identity; "A", "B", "C") Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) HPI (Time in hours at which systemic bacterial load was measured; "4" denotes 4 hours post-infection, "10" denotes 10 hours post-infection) FlyID (Identity number of individual fly for which systemic bacterial load was measured) Log2CFU (Log (base 2) transformed value of systemic pathogen load (CFU*Dilution factor) estimated for each individual fly) Tab 3. "P.rettgeri" 1\. Number of variables: 7 2\. Number of cases/rows: 289 3\. Variable List: Pathogen (Pathogen used to infect the hosts; "Pr" denotes P. rettgeri) Replicate (Replicate identity; "A", "B", "C") Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) HPI (Time in hours at which systemic bacterial load was measured; "4" denotes 4 hours post-infection, "10" denotes 10 hours post-infection) FlyID (Identity number of individual fly for which systemic bacterial load was measured) Log2CFU (Log (base 2) transformed value of systemic pathogen load (CFU*Dilution factor) estimated for each individual fly) #### File: README\_survival.txt **Description:** 1. Title of Dataset: "Effect of resource limitation on host post-infection survival and systemic pathogenic growth" 2\. Author Information A. Principal Investigator Contact Information Name: Prof. N. G. Prasad Institution: Indian Institute of Science Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India. Email: prasad@iisermohali.ac.in B. Associate or Co-investigator Contact Information Name: Aabeer Basu Institution: Indian Institute of Science Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India. Email: aabeerkbasu@gmail.com 3\. Duration of data collection: September 2018 - February 2019 4\. Geographic location of data collection: Mohali, Punjab, India 5\. Information about funding sources that supported the collection of the data: IISER Mohali, Govt. of India. ##### Variables * DATA & FILE OVERVIEW File Name:"survival_data.xlsx" Note: This file contains five separate tabs, each containing data from from experiments using a individual pathogen: Tab 1. "E.c.carotovora" Tab 2. "E.faecalis" Tab 3. "P.entomophila" Tab 4. "P.rettgeri" Tab 5. "S.succinus" Tab 1. "E.c.carotovora" 1\. Number of variables: 6 2\. Number of cases/rows: 1439 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_status (Denotes whether the host was subjected to infection or was sham-infected; "Ec" denotes infected hosts, "Sham" denotes sham-infected hosts) Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Tab 2. "E.faecalis" 1\. Number of variables: 6 2\. Number of cases/rows: 1430 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_status (Denotes whether the host was subjected to infection or was sham-infected; "Ef" denotes infected hosts, "Sham" denotes sham-infected hosts) Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Tab 3. "P.entomophila" 1\. Number of variables: 6 2\. Number of cases/rows: 1422 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_status (Denotes whether the host was subjected to infection or was sham-infected; "Pe" denotes infected hosts, "Sham" denotes sham-infected hosts) Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Tab 4. "P.rettgeri" 1\. Number of variables: 6 2\. Number of cases/rows: 1435 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_status (Denotes whether the host was subjected to infection or was sham-infected; "Pr" denotes infected hosts, "Sham" denotes sham-infected hosts) Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Tab 5. "S.succinus" 1\. Number of variables: 6 2\. Number of cases/rows: 1440 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_status (Denotes whether the host was subjected to infection or was sham-infected; "Ss" denotes infected hosts, "Sham" denotes sham-infected hosts) Mating_status (Denotes whether the host was mated before being infected/sham-infected; "Virgin" denotes hosts not allowed to mate, "Mated" denotes mated hosts) Nutrition_status (Denotes whether the host was subjected to starvation or not; "Fed" denotes hosts with access to food, "Sratved" denotes hosts subjected to starvation) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data)