Skip to main content
Download PDF

Effects of heat tolerance on the gut microbiota of Sarcophaga peregrina (Diptera: Sarcophagidae) and impacts on the life history traits

Parasites & Vectors volume 16, Article number: 364 (2023) Cite this article

Abstract

Background

Heat tolerance is a distinct abiotic factor affecting the distribution and abundance of insects. Gut microbiota can contribute to host fitness, thereby increasing resistance to abiotic stress conditions. In this study, Sarcophaga peregrina is closely associated with human life in ecological habits and shows remarkable adaptability to daily and seasonal temperature fluctuations. To date, the role of gut microbiota in S. peregrina response to heat stress and its influence on the host phenotypic variability remain poorly studied.

Methods

We exposed S. peregrina to heat stress at 40 °C for 3 h every day throughout the developmental stages from newly hatched larva to adult, after which gut DNA was extracted from third-instar larvae, early pupal stage, late pupal stage, and newly emerged adults, respectively. Then, 16S rRNA microbial community analyses were performed.

Results

Firstly, we analyzed whether heat stress could have an impact on the life history traits of S. peregrina and showed that the growth rate of larvae was higher and the developmental time was significantly shorter after heat stress. We then proposed the role of the gut microbiota in the heat tolerance of S. peregrina, which indicated that the bacterial abundance and community structure changed significantly after heat tolerance. In particular, the relative abundance of Wohlfahrtiimonas and Ignatzschineria was higher in the third-instar larval larvae; the former increased and the latter decreased significantly after heat stress. To further explore the effect of disturbing the microbial community on thermotolerant phenotype, newly hatched larvae were fed with amikacin under heat stress, which indicated that the larval length and the whole developmental cycle was significantly shorter.

Conclusion

This study indicated that Wohlfahrtiimonas and Ignatzschineria should play an important role in the post-feeding stage under heat stress, but further study is still needed. In general, heat tolerance can affect the gut microbial community structure, which in turn affects the fitness of the host.

Graphical Abstract

Background

Temperature is the most significant abiotic factor affecting the behavior, life history, physiology, abundance, and distribution of organisms [1]. Insects drive terrestrial ecosystems, and their biology is closely related to ambient temperatures, as they are ectotherms [2]. Temperature fluctuates in the natural environment, and the effects of this variation have been acknowledged in fields as diverse as thermal tolerance physiology, biological control, insect-mediated pollination, disease vector biology, forensic entomology, and simulated climate warming studies [3,4,5]. In recent years, global warming has led to a substantial increase in the occurrence of extreme heat [6, 7]. In many parts of China, maximum daily temperatures often exceed 40 °C for several hours during the summer [8, 9], posing an extreme threat to the fitness of insects, as this negatively affects insects’ behavior, development, survival, reproduction, and offspring fitness [1, 2]. Insects have an optimal temperature range within which their biological functions are most adaptable, and they may suffer physiological losses that reduce their performance under supra-optimal temperatures. For example, the longer-term effects of heat stress significantly affected the survival, development, and fecundity of the whitefly Bemisia tabaci biotype B [10]. It was also reported that he pupae of Drosophila melanogaster failed to emerge as adults after heat stress [11], while heat and cold stress induced male sterility in Drosophila buzzatii [12]. In transgenic D. melanogaster, the proportion of eggs laid by females was significantly reduced after heat stress, suggesting that high temperature has an effect on the insects' offspring [13].

Generally, temperature can affect the composition and diversity of gut microbiota and alter the functional relationship between the host and gut microbiota, which will influence phenotypic variability such as behavioral and life history traits exhibited by the host [14,15,16]. The ongoing climate change is expected to impose strong selection pressures on the heat tolerance of ectotherms [17], and the gut microbiota can promote thermal tolerance of hosts [18]. Insect midguts provide a favorable environment for microbial colonization, and the midgut bacteria play an important role in the phenotype and fitness of the host [19, 20]. Indeed, recent studies have explored the impact of temperature on the gut microbiota of ectotherms, indicating that the microbiota is sensitive to ambient temperature [21, 22]. For example, D. melanogaster reared at low temperature (13 °C) showed the highest abundance of Wolbachia in the gut, while that reared at high temperature (31 °C) had the highest abundance of Acetobacter (Proteobacteria); in addition, isolating bacterial suspensions from flies reared at low temperatures and then feeding newly fleeced flies resulted in altered heat tolerance in the recipient flies. However, we were not able to link this directly to a change in the host bacterial composition [23]. It was subsequently demonstrated that D. melanogaster acclimated in warm conditions showed a higher abundance of Acetobacter bacteria and a lower abundance of Leuconostoc bacteria (Firmicutes) relative to cold-acclimated flies [24]. Furthermore, recent studies have shown that the diversity and richness of their gut microbiota were decreased when hosts were exposed to higher-temperature conditions [18, 23]. However, the effects of heat stress on the gut microbial community of dipteran flies have been rarely studied.

Therefore, it is worth exploring the relationship between temperature and the gut microbiota in flies to gain a better understanding of how ectotherm species respond to thermal challenges and adapt to new environments. In this study, Sarcophaga peregrina was selected (Additional file 1: Fig. S1), which is a member of the Sarcophagidae family (known as flesh fly). This family comprises approximately 3000 described species worldwide, and is closely associated with human life in ecological habits [25]. Sarcophaga peregrina is one of the most common flesh flies, with widespread distribution from tropical to subtropical areas of Palearctic, Oriental, and Oceanian regions. The reproductive cycle of S. peregrina mostly comprises three definite stages: larva, pupa, and adult. It is well known for adopting the reproductive strategy of ovoviviparity (or ovolarviparity) [26, 27], which can serve as a key indicator in forensic investigations [28], especially in post-mortem interval (PMI) estimation. This method mainly relies on calculating the development time of immature flies that colonize on the decomposed corpses [29], where temperature is the most crucial parameter affecting the development time of flies [26]. So far, most of the developmental time data have been collected at different constant temperatures, but the temperature of the natural environment is never constant [30]. The development time of flies is significantly different under constant temperature versus fluctuating temperature, so neglecting this factor can lead to significant misestimation of the PMI in forensic investigations [31].

As mentioned above, S. peregrina shows a prominent capacity for adaptation to daily and seasonal temperature fluctuations and can survive several hours under high temperature. To date, the role of the gut microbiota in S. peregrina response to heat stress and its influence on the host phenotypic variability remain poorly studied. In a previous study, we published the annotated chromosome-level genome of S. peregrina for the first time, where the availability of the genome (National Center for Biotechnology Information [NCBI] accession no. JABZEU000000000) allowed us to identify potential candidate genes as the reference genome [27]. Here, 40 °C was selected as the optimal temperature of heat stress based on preliminary experiments. We exposed S. peregrina to heat stress at 40 °C for 3 h every day throughout the developmental stages from newly hatched larva to adult, and a control group was reared at a constant temperature of 25 °C. The aim of this study was to elucidate the effects of heat stress on the mil community and life history traits of S. peregrina.

Methods

Cultivation of S. peregrina and thermal treatments

Adult specimens of S. peregrina were trapped with pork lung bait in Changsha, Hunan Province, China, employing pork lung as a medium for larviposition and larval rearing. In this study, five lines were established and inbred for six generations to reduce genetic variability. In each generation, mating pairs of adults from each line were kept at 25 ± 1 °C and 70 ± 5% relative humidity with a photoperiod regime of 12:12 h light/darkness in an artificial climate chamber (GPL-250A, Tianjin Laboratory Instrument Equipment Co. Ltd., Tianjin, China). Afterwards, the newly hatched larvae were divided into two groups, which were fed at 25 °C and under heat stress at 40 °C for 3 h every day until they emerged as adults, respectively. The sampled larvae were observed to determine the instar based on the number of clefts in the posterior spiracle. The larval stage includes the first, second, and third instar. The third-instar individuals that just jumped into sand were defined as the post-feeding stage. The post-feeding larvae eventually reached metamorphosis, referred to as the pupal period, and finally emerged as adults. Larvae were collected every 8 h until adult emergence (see Additional file 1: Fig. S1). Three replicates were performed.

The duration and intensity of heat stress were based on the duration and intensity of high temperatures in summer, which usually involve a few hours of particularly high temperatures in central China (max temperature 40 °C for approximately 3 h per day for 20 consecutive days) [32]. Thus, the treatments were designed to examine the effects of high temperature (40 °C) on life history parameters using periods of exposure of 3 h per day. Control groups were kept at a constant temperature of 25 °C to allow normal development. To further explore the effects of gut microbiota on the heat tolerance of S. peregrina, amikacin was selected as an antibiotic that interferes with the gut bacteria of S. peregrina, and was mixed uniformly into the minced meat. Those reared with amikacin-added minced pig lung were recorded as the AM group, and those fed minced meat without amikacin were designated as the CL group (see Additional file 2: Table S1). The larval feeding substrates were placed in the experimental group (heat stress at 40 °C).

16S rRNA microbial community analysis

A total of 240 samples were collected in the control group (25 °C) and the experimental group (heat stress at 40 °C), including third-instar larvae in the control group (L3, n=30) and the experimental group (EL3, n=30), as well as early pupal stage in the control group (P1, n=30) and the experimental group (EP1, n=30), late pupal stage in the control group (P4, n=30) and the experimental group (EP4, n=30), newly emerged adults in the control group (A0, n=30) and the experimental group (EA0, n=30). Ten samples were pooled into a biological replicate. Three biological replicates of each group were performed. Total DNA was extracted from pooled guts utilizing the MagPure Soil DNA KF Kit (Magen, Guangdong, China) following the manufacturer's protocol. Polymerase chain reaction (PCR) amplification of the V3–V4 region of the 16S ribosomal RNA (rRNA) gene was selected using the universal bacterial primers (343F: 5′-TACGGRAGGCAGCAG-3′; 798R: 5′-AGGGTATCTAATCCT-3′) [33]. The PCR products were purified using AMPure XP beads (Beckman Coulter, Inc., USA) and quantified with the Qubit dsDNA [double-stranded DNA] Assay Kit (Life Technologies). Sequencing was performed on an Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA).

Paired-end reads were filtered using DADA2 with the default parameters of QIIME 2 [34, 35]. The representative read of the amplicon sequence variant (ASV) was selected using the QIIME 2 package, and then annotated and blasted against the SILVA database v138 [35, 36]. Alpha diversity and beta diversity were used for estimating the microbial diversity, and were determined using QIIME software. The microbial diversity in samples was estimated using the alpha diversity including the Chao1, Goods_coverage, Shannon, Observed_species, Simpson, and PD_whole_tree indices. The Bray–Curtis distance matrix performed in the R package was used for Bray–Curtis principal coordinates analysis (PCoA) to estimate the beta diversity. Then we further analyzed the significant differences between different groups using analysis of variance (ANOVA) and Kruskal–Wallis statistical tests. The 16S rRNA sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Results

Effect of heat tolerance on the life history traits

In the control group and the heat tolerance group, there were significant differences in different developmental stages, including larval (first, second, third, and post-feeding stage), pupal stage, emergence time, and total development time, especially in the third instar (see Additional file 2: Table S2). The developmental time for larvae in the heat stress group was about 25 h shorter than that for the control group, while the whole developmental time for the experimental group was about 73 h shorter than that for the control group. Furthermore, the emergence rate decreased significantly after exposure to heat stress, indicating that heat stress had a significant effect on the development of S. peregrina.

Based on the statistical analysis of the developmental time mentioned above, this study analyzed the nonlinear regression equation of larval body length and development time (see Additional file 2: Table S3), which showed that the correlation coefficient (R2) of the fitting equations of the two groups was greater than 0.99, P < 0.001. Meanwhile, the relationship between the larval body length and the development time was also determined (see Additional file 1: Fig. S2). The result showed that the body length of the first-, second-, and third-instar larvae increased continuously, and then body length decreased gradually due to the shortening at the post-feeding stage. Compared with the control group, the growth rate of larvae in the heat stress group was higher, and the development time was significantly shorter. The body length of larvae reached the peak earlier, and the maximum value of body length was significantly greater than that in the control group.

Effect of heat tolerance on the gut bacterial diversity

Raw reads ranged from 78,174 to 81,591, and clean tags ranged from 39,377 to 70,614 after quality control. Quality filtering and the removal of chimeric sequences yielded 38,232–68,459 valid tags (see Additional file 2: Table S4), indicating that the sequencing data is of high quality. The number of ASVs ranged from 53 to 689 in each sample (Fig. 1a). Heat stress affects the abundance of individual ASVs. A total of 5012 ASVs were identified; 364 ASVs were shared between the two groups, whereas 2294 ASVs were unique to the heat-stressed group and 2354 ASVs were unique to the non-stressed group (see Additional file 1: Fig. S3). The impact of heat stress led to a decrease in abundance of ASVs, among which the relative abundance of 19 ASVs increased significantly after heat stress, in particular two ASVs (ASV_68 and ASV_41) with the highest abundance, whereas 23 ASVs decreased in relative abundance, in particular ASV_15, ASV_18, ASV_17, ASV_35, ASV_32, ASV_37, ASV_42, and ASV_64 (Wilcoxon rank-sum test, *P < 0.05) (Fig. 1b).

Fig. 1
figure 1

Heat stress induced changes in the microbial communities. a The shared and unique number of ASVs in all samples. b The relative abundance of ASVs decreased significantly after heat stress (Wilcoxon rank-sum test, *P < 0.05). c The alpha diversity was determined by analysis of the Simpson index, with the lower index value in the experimental group indicating that heat stress can reduce the diversity of microbial communities. d The analysis of non-metric multidimensional scaling (NMDS) indicated significant differences in microbial communities in different developmental stages of S. peregrina after heat stress (R2 = 0.58153, P < 0.001)

Full size image

The rarefaction curve indicated that the sequencing data were sufficient for all samples, as well as visually showing differences in the richness of species between samples (see Additional file 1: Fig. S5). The species accumulation curve tends to flatten gradually, indicating that the sampling is sufficient, and the samples can reflect the richness of species. The rank abundance curve reflects the abundance and uniformity of species in the sample (see Additional file 1: Fig. S6). The alpha diversity was determined by analysis of the Chao1, Good's coverage, Shannon, observed species, Simpson, and PD [phylogenetic diversity] whole tree indices, where the lower index value in the experimental group indicated that heat stress can reduce the diversity of microbial communities. Furthermore, all index values decreased significantly in the late pupal period throughout the developmental cycle, indicating that changes in gut microbiota after heat stress may have a significant impact on the emergence as adults at the later stage of pupae (Fig. 1c and see Additional file 2: Table S5). This may shed light on why high temperatures lead to lower emergence rates from the perspective of microorganisms. Additionally, beta diversity is the degree of diversity between biological environments, that is, the comparison of differences between samples in different groups. These differences are compared based on the similarity of ASV sequences or the structure of the community. The results showed that there are significant differences in the distribution and structure of gut microbiota. Non-metric multidimensional scaling (NMDS) analysis indicated significant differences in microbial communities between the control group and treatment group, as well as differences at different developmental stages (R2 = 0.58153, P < 0.001) (Fig. 1d).

Effect of heat tolerance on the distribution of bacterial community

We investigated the effect of heat stress on the gut microbiota of S. peregrina, revealing that the bacterial composition responded very differently to exposure at 40 °C for 3 h every day, in comparison with the control. At the phylum level, the relative abundance of Firmicutes increased from 10.1 to 18.7% under heat stress, followed by Bacteroidota (from 17.0 to 21.1%). Proteobacteria dominated the bacterial community, with the relative abundance decreasing from 69.1 to 56.6%, followed by Actinobacteriota (from 14.4 to 1.0%). At the genus level, the relative abundance of Providencia, which dominated the bacterial community, decreased significantly from 32.8 to 9.1%. The relative abundance of Ignatzschineria decreased from 14.7 to 7.7%. The relative abundance of Proteus increased from 7.7 to 18.3%, followed by Wohlfahrtiimonas (from 3.3 to 14.0%), Muribaculaceae (from 3.5 to 7.5%), and Faecalibacterium (from 0.4 to 3.5%) (Fig. 2a).

Fig. 2
figure 2

a The relative abundance of microbial communities at the genus level. b Heatmap showing the relative abundance of top bacterial flora between the heat stress group and the control group analyzed by ANOVA at the phylum level. c Canonical correlation analysis (CCA) indicating the relationship between samples, environmental factors (heat stress), and microorganisms, in which the red arrows represent the effect of heat stress and the blue arrows represent different microorganisms. Different colored dots indicated samples from different groups. The angle between microorganisms and environmental factors indicates the positive and negative correlations between species and environmental factors (acute angle: positive correlation; obtuse angle: negative correlation; right angle: no correlation)

Full size image

Taxonomic view across life stages in response to heat tolerance

In order to further determine whether there were significant differences in different groups, at the phylum level, ANOVA showed that the relative abundance of Proteobacteria, Bacteroidota, and Actinobacteriota at different developmental stages of S. peregrina differed significantly under heat stress (Fig. 3 and see Additional file 2: Table S6), while the relative abundance of bacteria at different developmental stages, in particular Proteobacteria, Bacteroidota, and Actinobacteriota, was also demonstrated (Fig. 2b and see Additional file 1: Fig. S6). At the genus level, the relative abundance of Wohlfahrtiimonas, Ignatzschineria, Providencia, Myroides, and Pseudomonas differed significantly (see Additional file 2: Table S7). Furthermore, these results indicated that at different developmental stages under heat stress, there were significant differences in microbial communities. For example, the relative abundance of Wohlfahrtiimonas was higher in the third-instar larvae and increased significantly after heat stress. The relative abundance of Ignatzschineria was highest at the third-instar larval stage, decreased significantly after heat stress, and gradually decreased throughout the developmental cycle. Additionally, the relative abundance of Providencia was highest in the newly emerged adults but decreased significantly after exposure to heat stress (see Additional file 1: Fig. S7 and Additional file 2: Table S7). Canonical correlation analysis (CCA) further demonstrated the effects of heat stress on microbiota (Fig. 2c).

Fig. 3
figure 3

Circos map presenting the corresponding relationship between samples and the microbial community, reflecting the proportion of each dominant microbial community in different groups. The outer and inner circles of the left semicircles represent the relative abundance at the phylum level and the proportion in different groups. The outer and inner circles of the right semicircles represent the different groups and the proportion of species at the phylum level, respectively

Full size image

Effects of amikacin on the midgut microbes and life history traits

Amikacin is an aminoglycoside with strong action against aerobic Gram-negative bacteria. To further explore the effect of microbial community changes on life history traits, newly hatched larvae were fed with amikacin to disturb the gut microbiota, and the larval length in the AM group was significantly shorter than that in the CL group after entering the third-instar larval stage (Fig. 4a). Moreover, we investigated the effect of amikacin on the whole developmental cycle, demonstrating that the developmental time in the AM group was shorter than that in the CL group; in particular, the difference was more significant during the post-feeding stage (P < 0.01) (Fig. 4b and see Additional file 2: Table S8). The results confirmed that amikacin can be used to explore the effect of heat stress on larval gut bacteria, but more appropriate concentrations should be further investigated. Furthermore, it can be tentatively inferred that Gram-negative bacteria, which are the main targets of amikacin, play an important role in the resistance of larvae to the adverse effects caused by heat stress.

Fig. 4
figure 4

Effects of amikacin on the midgut microbes and life history traits. a The effects of amikacin on the length of larvae. The larval length in the AM group was significantly shorter than that in the CL group, after entering the third-instar larval stage. b The effect of amikacin on the whole developmental cycle, demonstrating that the developmental time in the AM group was shorter than that in the CL group

Full size image

Discussion

With the intensification of global warming, the fitness of insects has been seriously affected [24, 37]. As a holometabolous insect, S. peregrina undergoes larval–pupal metamorphosis to emerge into adults during the life cycle, and temperature is a key abiotic factor affecting metamorphosis [38, 39]. This study showed that heat stress had a significant effect on the life history traits of S. peregrina, in particular with respect to the developmental time and larval body length. Gut microbiota can promote the adaptation of host physiology, resulting in increased resistance under abiotic stressful conditions [14, 19, 21]. Insect midgut microbial symbionts have been considered as an integral component in thermal adaptation due to their differential thermal sensitivity. Altered midgut microbial communities can influence both insect physiology and competence for important vector-borne pathogens [19, 40]. Therefore, we hypothesized that significant changes in the midgut microbiota in S. peregrina are associated with heat stress. In this study, we investigated the physiological changes coupled with thermal resistance and life history traits of S. peregrina reared at high temperatures. The larva-to-adult developmental time was significantly affected by temperature exposure. The rate of development was relatively fast under heat stress at 40 °C compared with constant temperature at 25 °C. Moreover, we explored the relationship between the gut microbiota and the thermal physiology of S. peregrina. These results suggest that heat stress alters the composition and diversity of the gut microbiota. In general, temperature-induced changes in the gut microbiome may impact host fitness, including effects on colonization resistance in the gut, host energy and nutrient assimilation, and host life history traits [22]. Warm conditions lead to a decrease in ASV number (richness) and diversity of the gut microbiota in ectotherms [41, 42]. In this study, we found that the relative abundance of ASVs increased significantly after heat stress. Alpha diversity, determined by analysis of Chao1 and Shannon indices, indicated that microbial community abundance changed significantly after heat stress. PCoA indicated significant differences in microbial communities under heat stress as well. To further identify the specific bacterial communities of S. peregrina, Wilcoxon analysis showed that almost all of the top 10 bacterial genera in relative abundance decreased significantly after heat stress, indicating that such stress can negatively affect host fitness.

The composition of the gut microbiota varies substantially across invertebrate animal species but tends to display higher relative abundance of Proteobacteria than gut-microbiota composition in vertebrates. Variation in ambient temperature has been associated with changes in the composition of the gut microbiota in diverse invertebrate lineages [22]. In insects, increases in temperature have been associated with increased relative abundance of Proteobacteria. Developmental temperature has been shown to impact the composition of the gut microbiota of fruit flies, with higher temperatures leading to increased abundance of Acetobacter and Proteobacteria [23]. A previous study found that conventional flies are more tolerant to high temperatures than germ-free flies, suggesting that the gut microbiota has a positive influence on the heat tolerance of Drosophila subobscura [24]. This may be due to the effect of the gut microbiota on the nutritional status of the host, which in turn determines the growth and development of ectotherms [23, 43, 44]. In general, these studies have used thermal acclimation to evaluate changes in the gut microbiota composition, and it was found that at warm temperatures, bacteria of the phylum Firmicutes gradually decreased in ectotherms of vertebrates [41], whereas warm acclimatization led to an increase in the relative abundance of Proteobacteria in invertebrate ectotherms [23]. We found that in the dominant microbial community, the relative abundance of Firmicutes significantly increased after heat stress, followed by Bacteroidota dominating the bacterial community, and the relative abundance of Proteobacteria and Actinobacteriota decreased dramatically. The relative abundance of Wohlfahrtiimonas was higher in the third-instar larvae and increased significantly after heat stress. The relative abundance of Ignatzschineria was highest at the third-instar larval stage, decreased significantly after heat stress, and gradually decreased throughout the developmental cycle. Meanwhile, newly hatched larvae that were fed with amikacin to disturb the gut microbiota entered the post-feeding stage prematurely. The results indicated that Wohlfahrtiimonas and Ignatzschineria play an important role in the larva entering the feeding stage under heat stress [45], with the former positively correlated with heat stress and the latter negatively correlated. Additionally, the relative abundance of Providencia was highest in the newly emerged adults but decreased significantly after exposure to heat stress. These results are generally consistent with previous studies; for example, increased temperature led to the enrichment of specific taxa in Aedes aegypti, and the top five core phyla in relative abundance were Actinobacteria, followed by Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria [46].

Previous studies have demonstrated that bacterial diversity and abundance decline when hosts are exposed to a thermal environment, as temperature induces changes in the composition and diversity of the gut microbiota, which may have a dramatic effect on the phenotype and fitness of the host [47], suggesting that the gut microbiota is sensitive to ambient temperature. Indeed, thermal acclimation can affect the taxonomical abundance, diversity, and community structure of the gut microbiota of D. melanogaster, with higher temperatures leading to decreased abundance of Leuconostoc (Firmicutes), and increased abundance of Acetobacter bacteria (Proteobacteria) may be caused by providing more optimal growth conditions for acetic acid bacteria [23, 41]. Effects of temperature on the relative abundance of Proteobacteria lineages have also been observed in worms (Caenorhabditis elegans), where worms reared at higher temperatures displayed increased relative abundance of Agrobacterium and Proteobacteria, and a corresponding decrease in the relative abundance of Sphingobacterium, a genus of Bacteroidetes [48]. Consistent with previous studies on D. melanogaster, the composition and diversity of microbiota of flies developed at different temperatures were simple and consisted mainly of three genera: Acetobacter, Wolbachia, and Leuconostoc [49, 50]. In addition, D. melanogaster acclimated in warm conditions showed a higher abundance of Acetobacter and a lower abundance of Leuconostoc [23]. Here, we found that thermal acclimation led to an increase in the relative abundance of Proteus, which dominated the bacterial community, whereas the relative abundance of Providencia decreased significantly. Moreover, the higher abundance of Wolbachia in flies developed at higher temperatures supports a key involvement of temperature in regulating the presence of this genus in flies and suggests that Wolbachia have an impact on host temperature tolerance [23]. The influence of Wolbachia on the level and biosynthesis of octopamine depends on the endosymbiont genotype [51]. Moreover, the influence of the Wolbachia genotype on Drosophila survivability under heat stress correlates with changes in catecholamine (dopamine and octopamine) metabolism in the latter [52]. It is known that catecholamines are stress hormones in insects, which allows us to suppose that the influence of Wolbachia on host adaptability could be mediated through changes in the latter's catecholamine metabolism [52, 53].

Conclusions

In general, heat stress can have an impact on the life history traits of S. peregrina. We further found that heat tolerance can affect the gut microbial community structure, which in turn affects the fitness of S. peregrina. Wohlfahrtiimonas and Ignatzschineria should play an important role in the process of metamorphosis after heat stress. To further explore the effect of microbial community disturbance on thermotolerance phenotype, amikacin was fed to newly hatched larvae under heat stress, which indicated that the larval length and the whole developmental cycle were significantly shortened. However, 16S sequencing and interference verification were not performed for the intestinal flora of samples in the antibiotic feeding group under heat stress, and thus further study is still needed.

Availability of data and materials

Raw sequencing data of 16S rRNA have been deposited in the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) via accession numbers SRR17717693–SRR17717722 with the Bioproject ID PRJNA799843.

References

  1. Ma CS, Ma G, Pincebourde S. Survive a warming climate: insect responses to extreme high temperatures. Annu Rev Entomol. 2021;66:163–84. https://doi.org/10.1146/annurev-ento-041520-074454.

    Article  CAS  PubMed  Google Scholar 

  2. González-Tokman D, Córdoba-Aguilar A, Dáttilo W, Lira-Noriega A, Sánchez-Guillén RA, Villalobos F. Insect responses to heat: physiological mechanisms, evolution and ecological implications in a warming world. Biol Rev Camb Philos Soc. 2020;95:802–21. https://doi.org/10.1111/brv.12588.

    Article  PubMed  Google Scholar 

  3. Charabidze D, Hedouin V. Temperature: the weak point of forensic entomology. Int J Legal Med. 2019;133:633–9. https://doi.org/10.1007/s00414-018-1898-1.

    Article  PubMed  Google Scholar 

  4. Vasseur DA, DeLong JP, Gilbert B, Greig HS, Harley CD, McCann KS, et al. Increased temperature variation poses a greater risk to species than climate warming. Proc Biol Sci. 2014;281:20132612. https://doi.org/10.1098/rspb.2013.2612.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Colinet H, Sinclair BJ, Vernon P, Renault D. Insects in fluctuating thermal environments. Annu Rev Entomol. 2015;60:123–40. https://doi.org/10.1146/annurev-ento-010814-021017.

    Article  CAS  PubMed  Google Scholar 

  6. Overgaard J, Sørensen JG. Rapid thermal adaptation during field temperature variations in Drosophila melanogaster. Cryobiology. 2008;56:159–62. https://doi.org/10.1016/j.cryobiol.2008.01.001.

    Article  CAS  PubMed  Google Scholar 

  7. Meehl GA, Tebaldi C. More intense, more frequent, and longer lasting heat waves in the 21st century. Science. 2004;305:994–7. https://doi.org/10.1126/science.1098704.

    Article  CAS  PubMed  Google Scholar 

  8. Zhang W, Chang XQ, Hoffmann A, Zhang S, Ma CS. Impact of hot events at different developmental stages of a moth: the closer to adult stage, the less reproductive output. Sci Rep. 2015;5:10436. https://doi.org/10.1038/srep10436.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Terblanche JS, Hoffmann AA, Mitchell KA, Rako L, le Roux PC, Chown SL. Ecologically relevant measures of tolerance to potentially lethal temperatures. J Exp Biol. 2011;214:3713–25. https://doi.org/10.1242/jeb.061283.

    Article  PubMed  Google Scholar 

  10. Guo JY, Cong L, Wan FH. Multiple generation effects of high temperature on the development and fecundity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B. Insect Sci. 2013;20:541–9. https://doi.org/10.1111/j.1744-7917.2012.01546.x.

    Article  PubMed  Google Scholar 

  11. Mitchell HK, Lipps LS. Heat shock and phenocopy induction in Drosophila. Cell. 1978;15:907–18. https://doi.org/10.1016/0092-8674(78)90275-1.

    Article  CAS  PubMed  Google Scholar 

  12. Vollmer JH, Sarup P, Kaersgaard CW, Dahlgaard J, Loeschcke V. Heat and cold-induced male sterility in Drosophila buzzatii: genetic variation among populations for the duration of sterility. Heredity (Edinb). 2004;92:257–62. https://doi.org/10.1038/sj.hdy.6800405.

    Article  CAS  PubMed  Google Scholar 

  13. Silbermann R, Tatar M. Reproductive costs of heat shock protein in transgenic Drosophila melanogaster. Evolution. 2000;54:2038–45. https://doi.org/10.1111/j.0014-3820.2000.tb01247.x.

    Article  CAS  PubMed  Google Scholar 

  14. Broderick NA, Lemaitre B. Gut-associated microbes of Drosophila melanogaster. Gut Microbes. 2012;3:307–21. https://doi.org/10.4161/gmic.19896.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Hoye BJ, Fenton A. Animal host–microbe interactions. J Anim Ecol. 2018;87:315–9. https://doi.org/10.1111/1365-2656.12788.

    Article  PubMed  Google Scholar 

  16. Wernegreen JJ. Mutualism meltdown in insects: bacteria constrain thermal adaptation. Curr Opin Microbiol. 2012;15:255–62. https://doi.org/10.1016/j.mib.2012.02.001.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hoffmann AA, Sgrò CM. Climate change and evolutionary adaptation. Nature. 2011;470:479–85. https://doi.org/10.1038/nature09670.

    Article  CAS  PubMed  Google Scholar 

  18. Kokou F, Sasson G, Nitzan T, Doron-Faigenboim A, Harpaz S, Cnaani A, et al. Host genetic selection for cold tolerance shapes microbiome composition and modulates its response to temperature. Elife. 2018;7:e36398. https://doi.org/10.7554/eLife.36398.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dillon RJ, Dillon VM. The gut bacteria of insects: nonpathogenic interactions. Annu Rev Entomol. 2004;49:71–92. https://doi.org/10.1146/annurev.ento.49.061802.123416.

    Article  CAS  PubMed  Google Scholar 

  20. Douglas AE. The Drosophila model for microbiome research. Lab Anim (NY). 2018;47:157–64. https://doi.org/10.1038/s41684-018-0065-0.

    Article  PubMed  Google Scholar 

  21. Renoz F, Pons I, Hance T. Evolutionary responses of mutualistic insect-bacterial symbioses in a world of fluctuating temperatures. Curr Opin Insect Sci. 2019;35:20–6. https://doi.org/10.1016/j.cois.2019.06.006.

    Article  PubMed  Google Scholar 

  22. Sepulveda J, Moeller AH. The effects of temperature on animal gut microbiomes. Front Microbiol. 2020;11:384. https://doi.org/10.3389/fmicb.2020.00384.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Moghadam NN, Thorshauge PM, Kristensen TN, de Jonge N, Bahrndorff S, Kjeldal H, et al. Strong responses of Drosophila melanogaster microbiota to developmental temperature. Fly (Austin). 2018;12:1–12. https://doi.org/10.1080/19336934.2017.1394558.

    Article  PubMed  Google Scholar 

  24. Jaramillo A, Castañeda LE. Gut microbiota of Drosophila subobscura contributes to its heat tolerance and is sensitive to transient thermal stress. Front Microbiol. 2021;12:654108. https://doi.org/10.3389/fmicb.2021.654108.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Pape T. Catalogue of the Sarcophagidae of the World (Insecta: Diptera). Mem Entomol Int. 1996;8:1–558.

    Google Scholar 

  26. Wang Y, Wang JF, Zhang YN, Tao LY, Wang M. Forensically important Boettcherisca peregrina (Diptera: Sarcophagidae) in China: development pattern and significance for estimating postmortem interval. J Med Entomol. 2017;54:1491–7. https://doi.org/10.1093/jme/tjx139.

    Article  CAS  PubMed  Google Scholar 

  27. Ren L, Shang Y, Yang L, Wang S, Wang X, Chen S, et al. Chromosome-level de novo genome assembly of Sarcophaga peregrina provides insights into the evolutionary adaptation of flesh flies. Mol Ecol Resour. 2021;21:251–62. https://doi.org/10.1111/1755-0998.13246.

    Article  CAS  PubMed  Google Scholar 

  28. Goff ML, Omori AI, Goodbrod JR. Effect of cocaine in tissues on the development rate of Boettcherisca peregrina (Diptera: Sarcophagidae). J Med Entomol. 1989;26:91–3. https://doi.org/10.1093/jmedent/26.2.91.

    Article  CAS  PubMed  Google Scholar 

  29. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall MJ. Best practice in forensic entomology—standards and guidelines. Int J Legal Med. 2007;121:90–104. https://doi.org/10.1007/s00414-006-0086-x.

    Article  PubMed  Google Scholar 

  30. Shang Y, Ren L, Yang L, Wang S, Chen W, Dong J, et al. Differential gene expression for age estimation of forensically important Sarcophaga peregrina (Diptera: Sarcophagidae) Intrapuparial. J Med Entomol. 2020;57:65–77. https://doi.org/10.1093/jme/tjz137.

    Article  CAS  PubMed  Google Scholar 

  31. Niederegger S, Pastuschek J, Mall G. Preliminary studies of the influence of fluctuating temperatures on the development of various forensically relevant flies. Forensic Sci Int. 2010;199:72–8. https://doi.org/10.1016/j.forsciint.2010.03.015.

    Article  PubMed  Google Scholar 

  32. Chen H-S, Zheng X-W, Luo M, Guo J-Y, Luo Y-H, Zhou Z-S, et al. Effects of high temperature on body size and weight of Ophraella communa. Biocontrol Sci Tech. 2014;24:882–90. https://doi.org/10.1080/09583157.2014.902426.

    Article  Google Scholar 

  33. Nossa CW, Oberdorf WE, Yang L, Aas JA, Paster BJ, Desantis TZ, et al. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World J Gastroenterol. 2010;16:4135–44. https://doi.org/10.3748/wjg.v16.i33.4135.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3. https://doi.org/10.1038/nmeth.3869.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–7. https://doi.org/10.1038/s41587-019-0209-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl Acids Res. 2013;41:D590–6. https://doi.org/10.1093/nar/gks1219.

    Article  CAS  PubMed  Google Scholar 

  37. Huey RB, Kearney MR, Krockenberger A, Holtum JA, Jess M, Williams SE. Predicting organismal vulnerability to climate warming: roles of behaviour, physiology and adaptation. Philos Trans R Soc Lond B Biol Sci. 2012;367:1665–79. https://doi.org/10.1098/rstb.2012.0005.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Rolff J, Johnston PR, Reynolds S. Complete metamorphosis of insects. Philos Trans R Soc Lond B Biol Sci. 2019;374:20190063. https://doi.org/10.1098/rstb.2019.0063.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Truman JW, Riddiford LM. The origins of insect metamorphosis. Nature. 1999;401:447–52. https://doi.org/10.1038/46737.

    Article  CAS  PubMed  Google Scholar 

  40. Engel P, Moran NA. The gut microbiota of insects—diversity in structure and function. FEMS Microbiol Rev. 2013;37:699–735. https://doi.org/10.1111/1574-6976.12025.

    Article  CAS  PubMed  Google Scholar 

  41. Bestion E, Jacob S, Zinger L, Di Gesu L, Richard M, White J, et al. Climate warming reduces gut microbiota diversity in a vertebrate ectotherm. Nat Ecol Evol. 2017;1:161. https://doi.org/10.1038/s41559-017-0161.

    Article  PubMed  Google Scholar 

  42. Li YF, Yang N, Liang X, Yoshida A, Osatomi K, Power D, et al. Elevated seawater temperatures decrease microbial diversity in the gut of Mytilus coruscus. Front Physiol. 2018;9:839. https://doi.org/10.3389/fphys.2018.00839.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Ridley EV, Wong AC, Westmiller S, Douglas AE. Impact of the resident microbiota on the nutritional phenotype of Drosophila melanogaster. PLoS ONE. 2012;7:e36765. https://doi.org/10.1371/journal.pone.0036765.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Henry Y, Colinet H. Microbiota disruption leads to reduced cold tolerance in Drosophila flies. Naturwissenschaften. 2018;105:59. https://doi.org/10.1007/s00114-018-1584-7.

    Article  CAS  PubMed  Google Scholar 

  45. Xu W, Wang Y, Wang YH, Zhang YN, Wang JF. Diversity and dynamics of bacteria at the Chrysomya megacephala pupal stage revealed by third-generation sequencing. Sci Rep. 2022;12:2006. https://doi.org/10.1038/s41598-022-06311-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Onyango GM, Bialosuknia MS, Payne FA, Mathias N, Ciota TA, Kramer DL. Increase in temperature enriches heat tolerant taxa in Aedes aegypti midguts. Sci Rep. 2020;10:19135. https://doi.org/10.1038/s41598-020-76188-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Alberdi A, Aizpurua O, Bohmann K, Zepeda-Mendoza ML, Gilbert MTP. Do vertebrate gut metagenomes confer rapid ecological adaptation? Trends Ecol Evol. 2016;31:689–99. https://doi.org/10.1016/j.tree.2016.06.008.

    Article  PubMed  Google Scholar 

  48. Berg M, Stenuit B, Ho J, Wang A, Parke C, Knight M, et al. Assembly of the Caenorhabditis elegans gut microbiota from diverse soil microbial environments. Isme j. 2016;10:1998–2009. https://doi.org/10.1038/ismej.2015.253.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Buchon N, Broderick NA, Lemaitre B. Gut homeostasis in a microbial world: insights from Drosophila melanogaster. Nat Rev Microbiol. 2013;11:615–26. https://doi.org/10.1038/nrmicro3074.

    Article  CAS  PubMed  Google Scholar 

  50. Chandler JA, Lang JM, Bhatnagar S, Eisen JA, Kopp A. Bacterial communities of diverse Drosophila species: ecological context of a host–microbe model system. PLoS Genet. 2011;7:e1002272. https://doi.org/10.1371/journal.pgen.1002272.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rohrscheib CE, Bondy E, Josh P, Riegler M, Eyles D, van Swinderen B, et al. Wolbachia influences the production of octopamine and affects Drosophila male aggression. Appl Environ Microbiol. 2015;81:4573–80. https://doi.org/10.1128/aem.00573-15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Gruntenko N, Ilinsky YY, Adonyeva NV, Burdina EV, Bykov RA, Menshanov PN, et al. Various Wolbachia genotypes differently influence host Drosophila dopamine metabolism and survival under heat stress conditions. BMC Evol Biol. 2017;17:252. https://doi.org/10.1186/s12862-017-1104-y.

    Article  CAS  PubMed  Google Scholar 

  53. Gruntenko NE, Adonyeva NV, Burdina EV, Karpova EK, Andreenkova OV, Gladkikh DV, et al. The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions. Biol Open. 2016;5:1706–11. https://doi.org/10.1242/bio.022038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We greatly thank Prof. Lushi Chen (Guizhou Police College) for species identification. We are also very grateful to the editors and reviewers for valuable suggestions, which greatly improved the quality of manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82072114), Natural Science Foundation of Hunan Province (Nos. 2023JJ40797 and 2023JJ40794), and Changsha Municipal Natural Science Foundation (No. kq2208301).

Author information

Authors and Affiliations

  1. Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, Hunan, China

    Lipin Ren, Xiangyan Zhang, Fengqin Yang, Ngando Fernand Jocelin, Yanjie Shang & Yadong Guo

  2. OE Biotech Co. Ltd, Shanghai, China

    Qing Wang

  3. Health Law Research Center, School of Law, Central South University, Changsha, Hunan, China

    Zhuoying Liu

Authors
  1. Lipin Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiangyan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fengqin Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ngando Fernand Jocelin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanjie Shang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhuoying Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yadong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YG, ZL and LR designed the research. LR, XZ and FJ performed the experiments. LR, FY, YS and QW analyzed the data. YG and ZL contributed technical support. LR wrote the manuscript; YG and ZL revised the manuscript; all authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Zhuoying Liu or Yadong Guo.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1. Fig. S1

: A female adult specimen of S. peregrina. Fig. S2: The relationship between the larval body length and the developmental time is shown. Fig. S3: This study showed the shared and unique number of ASVs in the control group and heat stress group, indicating that heat stress induced changes in the abundance of individual ASVs. Fig. S4: Rarefaction curves were used to calculate indices based on Good's coverage. Fig. S5: The rank abundance curve reflects the abundance and uniformity of species in the sample. Fig. S6: The relative abundance of top bacterial flora between the heat stress group and the control group analyzed by ANOVA at the phylum level. Fig. S7: The relative abundance of top bacterial flora between the heat stress group and the control group analyzed by ANOVA at the genus level.

Additional file 2. Table S1

: Feeding substrate preparation. Table S2: Development time of S. peregrina under heat stress (h) Mean (± SD). Table S3: The nonlinear regression equation of larval body length and development time. Table S4: Summary statistics of tags during developmental stages in response to heat stress. Table S5: The effects of heat stress on bacterial alpha diversity of S. peregrina across life stages. Table S6: The relative abundance of top bacterial flora between the heat stress group and the control group analyzed by ANOVA at the phylum level. Table S7: The relative abundance of top bacterial flora between the heat stress group and the control group analyzed by ANOVA at the genus level. Table S8: Mean (± SD) development duration (h) with different treatment methods.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ren, L., Zhang, X., Yang, F. et al. Effects of heat tolerance on the gut microbiota of Sarcophaga peregrina (Diptera: Sarcophagidae) and impacts on the life history traits. Parasites Vectors 16, 364 (2023). https://doi.org/10.1186/s13071-023-05973-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13071-023-05973-0

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us