The extraembryonic serosa is a frontier epithelium providing the insect egg with a full-range innate immune response

  1. Chris G C Jacobs
  2. Herman P Spaink
  3. Maurijn van der Zee  Is a corresponding author
  1. Leiden University, Netherlands
7 figures, 3 tables and 9 additional files

Figures

Counts of colony forming units (cfu's) after sterile and septic injury.

Green lines represent bacterial growth in wild-type eggs. Red lines represent bacterial growth in Tc-zen1 RNAi (serosa-less) eggs. Sterile injury did not introduce bacteria (lower lines: average of 2 cfu's found at t = 0 and an average of 5 cfu's found at t = 6). Septic injury introduced on average 53 bacteria into wild-type eggs and 49 into serosa-less eggs. These numbers increased to 747 ± 106 cfu's in wild-type eggs (green upper line) and to 7260 ± 1698 cfu's in serosa-less eggs (red upper line) at t = 6. This means that bacteria propagate twice as fast in serosa-less eggs (p < 0.01, as determined by a Pearson's chi-square test). Suspensions of 10 eggs were used per LB agar plate (see ‘Materials and methods’), and 10 plates were analyzed per treatment and time point, giving rise to the error bars presented in the graph (standard error).

https://doi.org/10.7554/eLife.04111.003
Experimental setup.

(A) We collected eggs from wild-type, control RNAi, and Tc-zen1 RNAi beetles overnight. These eggs were incubated for 24 hr at 30°C to ensure development of the serosa. Eggs are then maximally 40 hr old, while total developmental time is close to 85 hr at 30°C. Eggs were pricked with a sterile needle (sterile injury), pricked with a mix of E. coli and M. luteus (septic injury), or remained untreated (naive). They were incubated for another 6 hr at 30°C before total RNA was extracted for RNAseq. To analyze the immune response, the transcriptomes of sterilely injured eggs and of septically injured eggs were compared to naive eggs. This was done for wild-type, control, and Tc-zen1 RNAi eggs. (B) We collected three biological samples for each combination of egg-type (wild-type, control RNAi, or Tc-zen1 RNAi) and treatment (naive, sterile injury, or septic injury) giving a total of 27 biological samples.

https://doi.org/10.7554/eLife.04111.004
Types of genes that are differentially regulated.

(A) Significantly over-represented GO-terms among the genes induced in wild-type eggs after septic injury (p < 0.001). (B) Significantly over-represented GO-terms among the genes induced in control RNAi eggs after septic injury (p < 0.001). These categories indicate that the detected differential regulation does not result from artefacts induced by treatments (such as death or delayed development) and show that Tribolium eggs display an elaborate immune response.

https://doi.org/10.7554/eLife.04111.005
Immune-responsive genes in wild-type, control, and Tc-zen1 RNAi eggs.

(A) Schematic representation of the immune signaling pathways in Tribolium as described in Zou et al. (2007). Significantly induced genes after septic injury in wild-type or control RNAi eggs are indicated in green; significantly repressed genes after septic injury in wild-type or control RNAi eggs are indicated in red. Genes not differentially expressed are black. The size of the gene names represents the fold change (small = 1.5- to 10-fold, medium = 10- to 500-fold, large = 500 + fold expression). (B) Venn diagram showing the number of differentially expressed genes in septically injured eggs as compared to naive eggs (FDR < 0.01). In total, 538 genes are differentially expressed upon infection, of which 394 in wild-type eggs, 435 in control RNAi eggs, and only 57 in Tc-zen1 RNAi eggs. This means that Tribolium eggs display an extensive transcriptional response upon infection and that this response is largely abolished in eggs without a serosa.

https://doi.org/10.7554/eLife.04111.007
RT-qPCR verification of immune gene expression.

The expression levels of several immune genes was verified by RT-qPCR. Expression shown relative to the expression in naive eggs, the mean fold change of the biological replicates (based on two technical replicates) is plotted and error bars show the standard error. Black bars represent expression after sterile injury, white bars represent expression after septic injury. Expression levels measured by RT-qPCR show very similar results as the expression levels measured by RNAseq (See Supplementary file 2). (A) PGRP-LC, (B) SPH-H57, (C) SPH-H70, (D) cSP-P8, (E) serpin24, (F) serpin26, (G) toll3, (H) TC004646, (I) TC007763, (J) TC007858, (K) TC008806, (L) TC015479. See ‘Materials and methods’ for experimental details.

https://doi.org/10.7554/eLife.04111.009
In situ hybridization showing expression of AMP genes in the serosa upon septic injury.

(AF) Thaumatin1 in situ hybridization. (A) Superficial view. Thaumatin1 is expressed around the site of injury (asterix). Brown melanisation is observed around the site of injury. (A′) Magnification of the expression area shown in (A). Asterix marks the site of injury. (B) DAPI counterstaining of the same egg as in (A). The large polyploid serosal nuclei can be distinguished from the oversaturated DAPI signal from the germ-band. Head lobes to the left. (B′) magnification of (B). (C) Overlay of the in situ hybridization shown in A and the DAPI staining shown in (B). The thaumatin1 expression associates with the large polyploid serosal nuclei and is not found in the embryo proper. (C′) Magnification of the expression area shown in (C). (D) Focal plane through the egg. Thaumatin1 is expressed in a thin outer layer at the surface of the egg. (D′) Magnification of the expression area shown in (D). (E) DAPI staining of the same egg shown in (D). The embryo is brightly visible. Head to the left. (E′) Magnification of E. (F) Overlay of the in situ hybridization shown in D and the DAPI staining shown in (E). (F′) Magnification of the expression area. (G) Attacin1 in situ hybridization. Brown melanisation is visible around the site of injury (arrowhead). (G′) Magnification of the anterior region of the egg shown in (G). (H) DAPI staining of the same egg shown in G. The germ-band is brightly stained (head to the left) and the separate large serosal nuclei are visible. (H′) Magnification of the anterior of the egg shown in (H). (I) Overlay of the in situ hybridization shown in G and the DAPI staining shown in (H). Attacin1 is expressed in the large serosal cells covering the germ-band and is not expressed in the dense cells of the germ band. (I′) Magnification of the anterior of the egg shown in (I). The attacin1 staining associates with the large serosal nuclei.

https://doi.org/10.7554/eLife.04111.010
Constitutive expression of immune genes in the serosa.

(AC) Toll3 in situ hybridization. (A) Toll3 is expressed in the flat and thin serosal cells (partly detached from the egg) but also in the germ rudiment (head lobes to the right). (A′) Magnification of the area indicated with an arrow in (A). (B) DAPI staining of the same egg shown in (A). The bright staining of the germ-band can be distinguished from the large nuclei of the serosa. (C) Overlay of the in situ hybridization shown in A and the DAPI staining shown in (B). (C′) Magnification of (C). Toll3 is expressed in cells of the serosa. (DF) Scavenger receptor B5 in situ hybridization. (D) Scavenger receptor B5 shows expression in every serosal cell at the surface. (D′) Magnification of (D). (E) DAPI staining of the same egg shown in (D). The germ-band is brightly stained (head to the left) and the staining of the serosal nuclei is clearly visible when not overwhelmed by staining of the dense nuclei of the germ-band. (E′) Magnification of (E). The serosal nuclei are visible. Bright staining of the germ-band to the right. (F) Overlay of the in situ hybridization shown in D and the DAPI staining shown in (E). Scavenger receptor B5 expression follows the serosal nuclei and is not detected in the germ-band. (F′) Magnification of (F). Scavenger receptor B5 mRNA is detected around the large polyploid serosal nuclei and not around the dense nuclei of the germ rudiment.

https://doi.org/10.7554/eLife.04111.012

Tables

Table 1

Number of differentially expressed immune genes in Tribolium castaneum eggs

https://doi.org/10.7554/eLife.04111.006
Wild-type sterile injuryWild-type septic injuryControl sterile injuryControl septic injuryTc-zen1 sterile injuryTc-zen1 septic injury
Microbial recognition417260831000
Extracellular signal transduction and modulation276321033434104520
Intracellular transduction pathways (Toll/IMD/JNK/JAK-STAT)213222633221
Execution/stress1202021642475231
Total45862165710722313972
updownupdownupdownupdownupdownupdown
  1. Blue = induction, red = repression.

Table 2

Antimicrobial properties of known and potential new antimicrobial peptides in Tribolium castaneum.

https://doi.org/10.7554/eLife.04111.008
Gene IDMolecular weight (kDa)Peptide length (AA)Hydrophobic ratioNet chargeGlycine contentProline contentFold change wild-typeFold change control
Cecropin1/TC0004993.673158%+56%0%InfInf
Cecropin3/TC0005009.809043%+26%13%Inf49x
attacin2/TC00773815.8014537%+712%4%3098x2190x
Coleoptericin1/TC00509315.9914130%−19%7%2392x18067x
Defensin2/TC0105178.737950%+66%1%1183xInf
Defensin3/TC0124699.428350%+73%1%908xInf
attacin1/TC00773717.4916528%+918%3%869x3696x
TC00785820.1418235%011%3%484x54x
Defensin1/TC00625014.9113246%+114%3%187x1551x
TC01103612.8910939%+132%6%138x11x
Coleoptericin2/TC00509615.9614130%−19%7%91x227x
TC01547913.0012042%+65%1%80x26x
TC00776316.8715837%+46%17%47x67x
TC00464615.0413534%+27%7%40x29x
TC00880615.8314233%+210%2%31x37x
TC00933613.5013730%−439%2%15x7x
TC01456520.7317638%+172%2%14x9x
TC00103014.6213729%+910%12%9x16x
TC00178413.5415027%+743%2%8x6x
TC00547813.7012245%+104%1%6x7x
TC01561220.3218236%+76%6%6x2x
TC0079017.256425%+57%10%6x5x
TC01530419.3018038%+26%9%5xno hit
TC01173311.8910646%+32%0%5x5x
TC00337412.2212461%+31%9%2x9x
TC00855717.8217231%+218%0%3x5x
TC01575415.6914034%+54%7%2x2x
TC00043511.8410537%+55%0%2x2x
TC00909612.8411116%+169%6%2x2x
  1. In the table are known antimicrobial peptides and those proteins that show at least a twofold induction upon infection, they are smaller than 200 amino acids and are not negatively charged. TC009336 was included because of the high glycine content.

Table 3

Differentially regulated immune genes in naive wild-type eggs compared to naive Tc-zen1 RNAi eggs

https://doi.org/10.7554/eLife.04111.011
Gene IDDescriptionFold changeFDR adjusted p-valueGene IDDescriptionFold changeFDR adjusted p-value
Extracellular signal transduction and modulation
TC000247cSPH-H22.70<0.01TC005754serpin225.26<0.01
TC000248cSPH-H34.11<0.01TC006255serpin240.690.03
TC000249cSPH-H45.16<0.01TC011718serpin271.62<0.01
TC000740SPH-H179.28<0.01TC006726Spz43.00<0.01
TC000829SPH-H188.26<0.01TC013304Spz5122.56<0.01
TC007026cSPH-H7829.79<0.01Microbial recognition
TC012390SPH-H1291.60<0.01TC002789PGRP-LA3.950.02
TC000495cSP-P86.57<0.01TC014664TEP-B2.900.02
TC000497cSP-P104.50<0.01TC005976PSH3.43<0.01
TC000547SP-P132.41<0.01TC006978C-type lectin114.52<0.01
TC000635SP-P162.54<0.01TC013911C-type lectin 1318.21<0.01
TC004160cSP-P449.79<0.01Toll-signalling pathway
TC004624cSP-P520.52<0.01TC004438Toll32.28<0.01
TC004635cSP-P5351.56<0.01IMD-signalling pathway
TC005230cSP-P61250.00<0.01TC014708NFAT2.01<0.01
TC006033SP-P681.54<0.01Execution mechanisms
TC009090cSP-P912.80<0.01TC005375hexamerin20.38<0.01
TC009092cSP-P933.00<0.01TC005493Heme peroxidase 13.84<0.01
TC009093cSP-P9427.76<0.01TC015234Heme peroxidase 26.30<0.01
TC013277cSP-P1363.04<0.01TC010356Scavenger receptor-B130.600.03
TC013415SP-P14111.85<0.01TC015854Scavenger receptor-B21.91<0.01
TC000760serpin15.29<0.01TC014946Scavenger receptor-B529.29<0.01
TC005750serpin181.92<0.01TC000948Scavenger receptor-B6163.90<0.01
TC005752serpin202.30<0.01TC014954Scavenger receptor-B91.96<0.01
  1. SP = serine protease; SPH = non-catalytic serine protease; cSP = clip-domain serine protease.

Additional files

Supplementary file 1

Summary statistics for Tribolium castaneum transcriptome sequencing analysis.

https://doi.org/10.7554/eLife.04111.013
Supplementary file 2

Significantly differentially expressed immune genes in wild-type, control, and Tc-zen1 RNAi eggs.

https://doi.org/10.7554/eLife.04111.014
Supplementary file 3

Primers used for RT-qPCR.

https://doi.org/10.7554/eLife.04111.015
Supplementary file 4

DEseq output from wild-type eggs, sterile injury compared to naive eggs.

https://doi.org/10.7554/eLife.04111.016
Supplementary file 5

DEseq output from wild-type eggs, septic injury compared to naive eggs.

https://doi.org/10.7554/eLife.04111.017
Supplementary file 6

DEseq output from control RNAi eggs, sterile injury compared to naive eggs.

https://doi.org/10.7554/eLife.04111.018
Supplementary file 7

DEseq output from control RNAi eggs, septic injury compared to naive eggs.

https://doi.org/10.7554/eLife.04111.019
Supplementary file 8

DEseq output from Tc-zen1 RNAi eggs, sterile injury compared to naive eggs.

https://doi.org/10.7554/eLife.04111.020
Supplementary file 9

DEseq output from Tc-zen1 RNAi eggs, septic injury compared to naive eggs.

https://doi.org/10.7554/eLife.04111.021

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  1. Chris G C Jacobs
  2. Herman P Spaink
  3. Maurijn van der Zee
(2014)
The extraembryonic serosa is a frontier epithelium providing the insect egg with a full-range innate immune response
eLife 3:e04111.
https://doi.org/10.7554/eLife.04111