Immune defense in Drosophila melanogaster depends on diet, sex and mating status

Post-mating immunosuppression has been widely accepted as a female trait in Drosophila melanogaster. Our results challenge this notion by presenting a mating-immunity trade-off in males as well as in females. When inoculated with the fungal pathogen, Beauveria bassiana, both males and females die faster compared to inoculated virgins, and survival is lower when inoculated flies are continuously mated compared to a single day of mating. Past studies with Beauveria bassiana have shown females to be more susceptible to infection than males. Our results challenge this finding as well, showing that the direction of sexual dimorphism in immune defense depends on mating status, specific Beauveria bassiana strain, and fly genotype. Moreover, we show that survival after fungal infection is largely influenced by diet, and that post-infection dietary improvements can help enhance survival. Post-mating suppression in Drosophila survival of B. bassiana infection presents study opportunities with potential applications for biological control of insect vectors of human disease and insect crop pests.

120 surface of anesthetized flies. After the spray, flies from each treatment were moved to 121 separate acrylic cages (volume: 450 cm³), fed with a Petri dish of fly medium, and kept at 122~100% humidity for 24 hours. In high humidity conditions, fungal conidia germinate, and 123 the hyphae penetrate the insect cuticle, entering the hemocoel (18). After 24 hours, 124 humidity was reduced to 60% for the duration of each assay.
The model was split into three time intervals: 0-4 days, 4-9 days, and 9-12 days 257 after spray. Over these time intervals, the coefficient estimates were different: The model was split into three time intervals: 0-5 days, 5-8 days, and 8-12 days 279 after spray. Each time interval has a model formula given below. Over these time intervals, 280 the coefficient estimates were different: 286 bassiana infection (Fig 1). Among infected females, virgins survived better than mated and 287 cohabiting females in all time intervals (Fig 1, Fig S1, Table S3), and mated females 288 survived better than cohabiting females (Fig 1, Fig S1, Table S3). For males, the same 289 pattern was true in the 5-11 and 11-21 day intervals (Fig 1, Fig S1, Table S3). Even among 290 control (uninfected) females and males, virgins showed a significantly higher survival than 291 cohabiting flies in the 5-11 and 11-21 intervals (Fig 1, Fig S1, Table S3).

in both males and females.
294 Data from Experiment 1. Flies were sprayed with fungal suspension (n = 1658, solid lines) 295 or with a control, fungus-free suspension (n = 1626, dashed lines) at age 17 days from egg, 296 and survival was followed for 21 days. In both females (green) and males (red), mated flies 297 (which mated for 24 hours prior to the spray) had lower survival than virgin flies, and flies 298 that mated for longer than one day (cohabiting flies, which mated for 24 hours prior to 299 spray and then cohabited with the other sex after the spray) had lower survival than mated 300 flies (see Table S1 for statistical analysis). This figure shows model estimates for survival 301 proportions, which are obtained from the raw survival data ( Figure S1).  Table S4). The same trend was observed in virgin flies (p-307 value = 0.0016, Fig S1, Table S4). However, the trend was reversed in mated flies, where 308 the males survived better than females (p-value = 0.0084; Fig S1, Table S4).

309
310 Reproductive output is affected by infection and mating status (Experiment 1).

311
Mated and cohabiting females had offspring counts that declined with age over the 312 span of 21 days post inoculation (Fig 2A). As the flies aged, the fungal inoculated groups 313 showed lower offspring counts than the controls, as indicated by the p-value of <0.001 in 314 the interaction between days and treatment (Table S5). In the control groups, females that 316 the fungal inoculated groups, the cohabiting and single-day mated females had the same 317 numbers of offspring measured by counting pupal casing (Fig 2B).

360
Control flies survived better than inoculated flies regardless of diet (Fig 4). Flies 361 that received Glucose diets post-spray survived better after inoculation with B. bassiana 363 predominantly in male flies who survived better than females (Fig 4). Table S7 (Table S7). When flies were 371 given a Glucose diet post inoculation, regardless of whether the pre-inoculation diet was 372 Cornmeal or Glucose, males survived better than females in both the 3-9 and 9-12 day 373 intervals post inoculation (Table S7). Table S8 (Table S8).

389
In all dietary conditions, control males and females showed a higher survival than 390 the fungal inoculated males and females (Fig 5). Cornmeal fed flies who received 391 Cornmeal diet before and after the spray had the lowest post-inoculation survival (Fig 5).
392 Flies that were fed with cornmeal before spray, but yeast supplemented cornmeal after the 393 spray showed a rescue in survival in both males and females (Fig 5, Table S9). Flies fed 394 with yeast supplemented cornmeal before the spray and cornmeal after the spray showed 395 lower survival than flies that received yeast supplementation before and after inoculation 396 (Fig 5, Table S9). The highest survival was seen in flies that were given Glucose diets 397 before and after the spray, followed by the condition in which flies received yeast 398 supplemented cornmeal diet before and after the spray (Fig 5). Yeast supplementation 399 affected sexual dimorphism in surviving inoculation (Fig 5, Table S10). When yeast 400 supplementation was provided after inoculation, it ablated the sexual dimorphism in 401 survival of inoculation (Table S10). However, when yeast supplementation was provided 402 before inoculation, but not after, sexual dimorphism in survival was present in the 3-9 day 403 time intervals, similarly to the control Cornmeal diet (Table S10). On the Glucose diet, 404 males survived better than females in both the 3-9 day and 9-12 day time intervals (Table   405 S10). Under uninfected conditions, when yeast supplementation was provided before and 406 after the spray, male survival was negatively impacted relative to female survival (Table   407 S10).

423
Control flies survived better than inoculated flies regardless of diet and did not 424 present sexual dimorphism (Fig 6). The addition of yeast supplement after inoculation, 425 when done at the same dose as in Experiment 4 (1 g yeast/ 5mL DI water) improved 426 survival of both males and females relative to flies maintained on Cornmeal diets (Fig 6, 427 Table S11) similar to the results of Experiment 4. At half the amount of yeast supplement, 428 post inoculation survival was still improved relative to the Cornmeal-only diet, but survival 429 was still significantly lower than with the higher dose of yeast supplementation (Table   430 S11). When 1.5x the amount of yeast supplement was added relative to the amount in 432 dimorphism in survival post-inoculation was present in the 0-5 and 5-8 day intervals for 433 the Cornmeal-only and lowest level of yeast supplementation (Table S12), but the sexual 434 dimorphism was ablated in the 0-5 day interval at higher levels of yeast supplementation 435 (Table S12).