DOI: 10.5061/dryad.vmcvdnd4b
title: 'Disease-smart outcrossing can enhance individual fitness and increase survival via immune priming against pathogens: new approaches to strengthen genetic rescue efforts

description: We studied the potential of combining insect immune priming with the
 introduction of diverse migrants to safeguard individuals from an inbred
 population from disease as a technique for enhancing genetic rescue
 efforts. Immune priming in insects refers to the stronger immune response
 insects have against pathogens following exposure. This enhanced immunity
 can be passed on to offspring and holds promise for insect conservation
 efforts against diseases. We compared the fitness benefits of individuals
 from a small, inbred population with two treatments: the addition of
 genetically diverse migrants that had not been primed and the addition of
 immune-primed migrants. While both types of migrants enhanced
 reproduction, as in cases of genetic rescue, only primed migrants led to
 improved survival on exposure to a pathogen. Better immunity led to a
 trade-off with reproduction in immune-primed migrants, but this was not
 evident upon outcrossing with the target individuals, revealing synergies
 between hybrid vigor and immune priming. Given the demographic constraints
 and stochasticity that can exacerbate the effects of disease outbreaks in
 small populations, our results serve as a proof of concept for combining
 immune priming with assisted migration to offer a proactive strategy to
 mitigate disease impacts while enhancing genetic diversity.
description: <em><strong>Study
 system</strong></em><br>Our populations were derived
 from a genetically diverse stock comprising five lineages of <em>T.
 castaneum </em>(Durkee et al. 2024). The stock population, which we
 used for the initial experiments and migrants, was maintained at 2000
 individuals housed in replicate enclosures 4cm × 4cm × 6cm containing 30g
 of medium (95% wheat flour, 5% brewer’s yeast), at 31°C and 54 ± 10%
 relative humidity. For the proof-of-concept rescue experiment, we used a
 single bottlenecked population, as detailed below. We chose not to include
 multiple distinct bottlenecked populations, as our primary objective was
 to assess individual-level fitness and immune responses. 
 We used females in our experimental priming treatments (“maternal
 priming”). <em>Bacillus thuringiensis</em> culture for priming
 (and later test infections) was prepared from a −80°C glycerol stock
 (strain: tenebrionis, procured from Bacillus Genetic Stock Center, Ohio),
 cultured overnight in nutrient broth at 30°C until reaching an optical
 density of 0.95 at 600nm. After centrifugation, the pellet was resuspended
 in 1mL PBS. Bacteria were either heat-killed for priming or used live for
 larval infections. Heat-killed bacteria were adjusted to 1011cells/mL of
 PBS for priming and 107 cells/mL of PBS for live challenge. We ensured
 this step by plating the heat-killed bacteria on agar plates and
 incubating them overnight for confirmation. For
 priming, adult females were chilled on ice for ten minutes to reduce
 movement. Under a microscope, each female was pricked between the head and
 thorax using a 0.1-mm pin dipped in a heat-killed bacterial solution.
 Sham-primed individuals were pricked in the same location but with a pin
 dipped in PBS, to serve as a control for the handling and slight injury.
 Pricked females were allowed 48 hours to heal before use in
 experiments. <strong>Initial experiments to
 develop foundational data: </strong> In this set
 of experiments, we used individuals from genetically diverse stock
 populations. We confirmed successful immune priming by measuring the
 survival of primed individuals and their offspring against disease, we
 examined potential immunological mechanism of priming, and we evaluated
 the effects of priming on offspring performance to measure potential
 trade-offs. Overall, we addressed four questions (Q1-Q4).
 <em>(Q1) Does immune priming enhance survival against
 disease in primed individuals?</em> To
 investigate the effects of immune priming on survival following pathogen
 challenge, we subjected female pupae to three treatments: control
 (unprimed), sham-primed, and primed. We then evaluated their survival
 after exposure to <em>Bacillus thuringiensis</em>
 (Bt). To rear pupae for priming, we established 30
 replicate groups of 50 adult beetles from the diverse stock population.
 Each group was placed in a 4 × 4 × 6 cm enclosure (hereafter, a patch)
 with standard diet. These beetles were allowed to mate and oviposit for 24
 hours, after which we sieved out the adults, retained the eggs in the
 diet, and allowed them to develop. When they were at the pupal stage,
 females were separated out (by using genital papillae as a marker) for use
 in the priming experiment. These female pupae were given an ID based on
 their patch of origin and placed individually in wells of a 96-well plate
 containing standard diet. The females developed into adults, and they were
 randomly assigned to one of the three treatments: a) unprimed control, b)
 sham-primed, and c) primed. Females in the primed treatment received
 immune priming using the protocol described above. Each treatment was
 applied to 10 females per patch, for a total of 300 adult females per
 treatment (Figure 1). Ten days after priming, adults from all three
 treatments were challenged with live <em>Bacillus
 thuringiensis</em> (Bt) with a dose adjusted to 107 cells/mL of PBS.
 Individual survival was recorded on day seven, which corresponds to Bt
 clearance time.  We evaluated female survival following
 pathogen exposure using a generalized linear mixed-effects model (GLMM)
 with a binomial error distribution and logit link. Treatment was included
 as a fixed effect, while patch was incorporated as a random effect to
 account for potential variation among patches. We used ‘emmeans’ for
 pairwise comparisons. <em>(Q2). Does maternal
 priming enhance offspring survival against disease?</em>
 To test for benefits of maternal priming to offspring (called
 trans-generational immune priming) we applied the same three treatments
 (unprimed control, sham-primed, and primed) to a second set of 300 unmated
 females and placed them individually in wells of 96-well plates with diet.
 These females were not challenged with pathogens during their lifetime.
 Forty-eight hours following priming, one male was added to each well, and
 the pairs were allowed to mate and oviposit for a total of 48 hours. After
 mating, the adults were removed, and the number of larvae produced by each
 female was counted to measure reproductive output. Each larva was assigned
 unique ID based on their maternal origin. These larvae were used to
 address this research question (Q2) regarding trans-generational effects,
 as well as the following two questions (Q3 and Q4) as described below. To
 evaluate transgenerational effects of maternal priming (Q2), we compared
 survival of larvae from each maternal treatment following pathogen
 exposure (nunprimed = 100, nsham-primed =101, nprimed = 115). <br>We
 validated trans-generational immune priming by analyzing larval survival
 following pathogen exposure using a generalized linear mixed model with a
 logit link function. The model included treatment applied to mothers as a
 fixed effect and patch ID as a random effect. Maternal identity could not
 be included, as only one larva per female was used to evaluate
 survival. <em>(Q3). Does maternal priming alter
 production of hemocytes and melanization in
 offspring?</em> We measured hemocytes and the
 production of melanin following previously established protocols (Ghosh et
 al. 2023a, b), focusing on larvae from primed and unprimed mothers (n=20
 and 48 larvae, respectively, for hemocytes and n=20 for both treatments
 for melanization). Briefly, larvae were incised laterally, and hemolymph
 was collected into pre-chilled Eppendorf tubes. To count hemocytes,
 hemolymph was diluted 1:2 with PBS, and 5 μL of this mixture was loaded
 onto a Neubauer hemocytometer, then counted under a microscope (200x
 magnification) after 15 minutes (n= 50/treatment). To assess production of
 melanin, we measured phenoloxidase activity by centrifuging hemolymph (1:2
 with ice-cold PBS), adding the supernatant to a microplate well with 2 mM
 L-Dopa (substrate), and recording melanization spectrophotometrically at
 490 nm after 30 minutes. Immunological parameters were analyzed using a
 linear mixed model, with hemocyte count or degree of melanization as the
 dependent variable, treatment (applied to mothers) as a fixed factor, and
 patch of maternal origin as a random effect. To explore
 mechanisms underlying the protective effects of maternal priming that we
 report below, we also evaluated if the number of hemocytes or degree of
 melanization in siblings could explain survival following pathogen
 exposure in generalized linear mixed models with survival as the dependent
 variable, and either sibling hemocytes or melanization as predictors,
 including patch of maternal origin as a random effect.
 <em>(Q4). Does maternal priming lead to trade-offs in
 offspring development time, weight and reproduction in the absence of
 disease?</em> To evaluate if maternal immune
 priming leads to trade-offs for their offspring, we examined three traits,
 offspring development time (egg-pupa, nunprimed = 49, nsham-primed = 30,
 nprimed = 41), offspring weight on eclosion to adulthood (nunprimed = 21,
 nsham-primed = 49, nprimed = 49), and offspring reproduction. To measure
 reproduction, we allowed a subset of the offspring from treated mothers to
 develop to the adult stage and used them to set up single mating pairs
 (20-25 pairs/treatment) in plastic vials containing 5g of flour. After a
 48-hour oviposition period, we sieved the mating pairs out. Offspring from
 each pair were counted on day 35. We analyzed adult
 body weight, development time, and reproduction data using a linear mixed
 model. In this model, we considered maternal treatment as our fixed effect
 and patch of maternal origin as a random effect. <br>Rescue
 Experiment: Integrating primed migrants into genetic rescue. In this
 section, we addressed two major questions- 
 <em>(Q5) In the context of genetic rescue, can immune
 priming of migrants protect the following generation from disease? And
 (Q6) are there associated benefits or trade-offs with maternal priming in
 terms of development time or reproduction in the following
 generation.</em> Creation of the target groups
 and migrants for the rescue experiment<br>The target population was
 one of the populations from Durkee et al. (2024). Briefly, the target
 population was founded with 50 individuals, thus imposing a mild
 demographic bottleneck. It then was reared in a challenging environment
 that had deltamethrin, an insecticide added to the medium for eight
 generations. This event simulated a sudden environmental change and
 imposed a selective bottleneck. At the end of the Durkee et al. (2024)
 experiment, the population was maintained on standard diet (without
 pesticide) for seven additional generations during which population size
 was at least 500 individuals. This history of a demographic bottleneck and
 strong selection, with the environment changing to more than less
 challenging conditions is not unlike what populations experience in nature
 and provides a target population that should have reduced genetic
 variation. More importantly, this setup simulates real-world scenarios,
 such as declining populations in captivity or degraded habitats, where
 assisted migration may play a critical role in boosting population fitness
 and genetic diversity.<br>Our migrants were derived from the
 genetically diverse stock population described above. In preparation for
 our experiment, an independent population was initiated with 50
 individuals in 4 × 4 × 6-cm enclosures partially filled with 30 mL of
 flour and yeast medium and allowed to grow to a size of 500 individuals
 over the course of several generations. This represented an isolated
 healthy population (monitored though stable positive growth rate). We used
 females from this population to create primed migrants as described above,
 with additional females as unprimed migrants. In our
 genetic rescue experiment, we added these migrants to groups of target
 individuals. Because the target individuals were from a population that
 had passed through a bottleneck, outcrossing with migrants from a
 genetically diverse population is expected to increase fitness by masking
 deleterious mutations that lead to genetic load, as is typical in efforts
 at genetic rescue (Whiteley et al. 2015). Our goal was then to examine
 whether immune priming could provide further benefits to the following
 generation, through enhancing survival in the presence of a pathogen. As
 such, we implemented three treatments: (a) groups of target individuals
 without any migrants (no migrants), serving as the control, (b), groups of
 target individuals with migrants that had not been immune primed (unprimed
 migrants), and (c) groups of target individuals with primed
 migrants.  Replicate control groups were initiated with
 50 beetles from the target population stock. Groups receiving migrants (of
 either type) were initiated with 40 individuals from the target stock, to
 which 10 unmated migrant females were added (20% of the population) (n=
 50). Maintaining group size at 50 ensures that the effects of migrants on
 the following generation are primarily genetic rather than demographic
 (Hufbauer et al. 2015). Although the ideal proportion of migrants used by
 managers may depend upon population size, existing genetic diversity, and
 the specific objectives of the initiative, typically, genetic rescue
 endeavors, including translocation programs, incorporate an introduction
 of migrants ranging from 10% to 20% of the recipient population (Hogg et
 al. 2006; Whiteley et al. 2015).  These experimental
 groups were given 48 hours for mating and oviposition, after which adults
 were removed. Seventeen days post-oviposition, two larvae were randomly
 collected from each patch and subdivided into two groups to assess the
 following: 1) larval survival following exposure to the Bt pathogen, and
 2) development duration (n = 50 per treatment) (Q5). The remaining larvae
 were allowed to develop into adults, and the average offspring number was
 calculated (Q6), accounting for the larvae used for Q5
 assessments. We compared larval survival following
 pathogen exposure between the control groups, and the groups that received
 primed migrants and unprimed migrants using a generalized linear mixed
 model with a binomial error distribution. Data on development duration and
 offspring production were analyzed similarly. All data were tested for
 normality (Shapiro-Wilk) and homogeneity of variances (Levene’s test), and
 all statistical analyses were performed using R version 4.2.2 (2014) and
 IBM SPSS (2024). Graphical abstract and visual designs were created using
 BioRender (License: Ghosh, E. 2025).
description: # 'Disease-smart' outcrossing can enhance individual fitness and
 increase survival via immune priming against pathogens: new approaches to
 strengthen genetic rescue efforts Dataset DOI:
 [10.5061/dryad.vmcvdnd4b](10.5061/dryad.vmcvdnd4b) ## **Description of the
 data and file structure** This folder contains the data for the article:
 'Disease-smart' outcrossing can enhance individual fitness and
 increase survival via immune priming against pathogens: new approaches to
 strengthen genetic rescue efforts. Insect Conservation and Diversity. Data
 description:  1\) Within generation priming: Patch corresponds to one
 replication having 50 beetles in a box, treatments are control, sham
 control, and primed, status is either dead (0) or alive (1) 2\) Trans
 generational priming: Treatments are control, sham control, and primed.
 Patch is the specific box number from where the mom was selected for
 priming, Mom ID is given to the selected primed beetle from the specific
 patch, status is either dead (0) or alive (1) 3\) Immune status:
 Treatments are control, sham control, and primed. Patch is the specific
 box number from where the mom was selected for priming, Mom ID is given to
 the selected primed beetle from the specific patch, Hemocytes number is
 from the larvae.