Physiological characterization of the emergence from diapause: A transcriptomics approach

Abstract

Organisms inhabiting high-latitude environments have evolved adaptations, such as diapause to time reproduction and growth to optimize their survival. However, the physiological regulation of the timing of complex life histories is poorly understood, particularly for marine copepods, that diapause at depth. A member of the pelagic community of the sub-Arctic Pacific Ocean, Neocalanus flemingeri enters diapause in June. Egg production occurs in winter/spring. In order to characterize the transition from diapause to egg release, females were collected in late September from 400-700 m depth, incubated in the dark at 4-5 °C and sampled for RNASeq at weekly intervals. The diapause phenotype showed down-regulation of protein turnover and up-regulation of stress genes. Activation of the reproductive program was marked by the up-regulation of genes involved in germline development. Thereafter, progress through phases of oocyte development could be linked to changes in gene expression. At 5 weeks, females showed up-regulation of spermatogenesis, indicating that stored sperm had been in a quiescent stage and completed their maturation inside the female. Gene expression profiles provide a framework to stage field-collected females. The 7-week progression from diapause to late oogenesis suggests that females typically spawning in January initiated the reproductive program in November.

Figure 1

Morphological observations of live N. flemingeri adult females during experimental incubation. (A) N. flemingeri adult female from Wk1 post collection, showing no evidence of oogenesis but sign of mating with opaque gonopores and seminal receptacles. (B) Adult female from Wk4 showing grainy appearance and creamy coloration; evidence of some oocytes in the anterior divernticulum. (C) Adult female from Wk7 with oocytes accumulated in lateral oviducts.

Figure 2

Biological processes represented in the N. flemingeri gene expression response during the 7-week incubation period following collection of diapausing individuals from depth. (A) Pie chart shows proportion of GO terms represented among the DEGs identified for N. flemingeri adult females in comparisons between Wk0 and Wk1–Wk7 individuals. For the pie chart, DEGs identified in each comparison were obtained independently and annotated. Annotation results for these DEGs were similar across all pair-wise comparisons and were averaged for the pie chart. For each GO term the percentage refers to the ratio between the total number of DEGs annotated within the term and the total number of annotated DEGs obtained for each paired comparison. (B) List of enriched GO terms for up-regulated (no arrow) and down-regulated genes (down arrow) in N. flemingeri adult females for each pair-wise comparison. 11 GO terms were enriched and based on ontology these terms fell within two broad functional GO terms “reproductive process” (blue; GO:0000003) and “multicellular organismal process” (orange; GO:0032501).

Figure 3

N. flemingeri reproductive process (GO:0000003). Heat map for DEGs involved in reproductive process (blue color in Fig. 2A) identified in pairwise comparisons between Wk0 females and all other time points (Wk1–Wk7). Color-coding for each gene indicates the magnitude of differential expression (log₂ FC [Wk_i/Wk0]). Columns are ordered by time point from Wk1 to Wk7 as indicated by the labels (top). Genes were ordered by similarity of expression pattern as shown by the dendrogram (left).

Figure 4

Relative expression of selected N. flemingeri protein encoding-genes involved in germline development, oocyte differentiation (MTOC), late egg formation and maternal control. Left - Germline development: (A) Neurogenic delta, (B) Innexin-2, (C) Apoptosis regulator BAX. Center - Oocyte differentiation (MTOC): (C) Spire, (D) Bicaudal, (E) Cappuccino. Right - Late egg formation and maternal control: (G) Maternal protein tudor, (H) Maternal protein torso, (I) Lysosomal aspartic protease. For all protein-encoding genes, relative expression is given as RPKM (reads per kilobase per million mapped reads) averaged across six replicate females for each time point (Wk0–Wk7). Error bars are standard deviations of six biological replicates. “*” Indicates differential gene expression between the week sample and Wk0.

Figure 5

Relative expression of selected N. flemingeri protein encoding-genes involved in male signal. Relative expression for (A) Ago3, (B) ATP-dependent RNA helicase and (C) Testis serine protease. Relative expression is shown in RPKM as the average expression and standard deviation (error bar) of six adult females for each time point. “*” Indicates differential gene expression between the week sample and Wk0.

Figure 6

Cluster analysis of N. flemingeri females during diapause emergence. Dendrogram of all adult females (n = 48) from Wk0 to Wk7 obtained by hierarchical cluster analysis based on 9,579 genes annotated as being involved in reproductive process (GO:0000003). Major clusters (I to IV; solid line) and sub-groups (dashed lines) are shown. Each line represents a single adult female, which is color-coded by time point: Wk0 [red]; Wk1 [orange]; Wk2 [yellow]; Wk3 [green]; Wk4 [light blue]; Wk5 [dark blue]; Wk6 [pink] and Wk7 [dark purple]. Length of the y-axis indicates distance between clusters and individuals.

Figure 7

Diagrammatic representation of the proposed developmental progression in N. flemingeri females from migration to depth (>400 m) to egg release. Central panel with arrow denotes experimental period starting with collection (Wk0, September 20, 2015) and ending in Wk7 (November 5, 2015). Side bars (indicated by ovals) on left (June/July) and right (November) indicate stages prior and after the current experimental period (Wk0–Wk7). Left: late spring/early summer pre-adults CV migrated to 400 m or deeper, molted into into adults, mated, and females entered diapause. Timing based on ecological time series. Prior to diapause, males attach a spermatophore to the female, which is followed by sperm transfer into the spermatheca, and removal of spermatophore. Center: developmental progression between Wk0 and Wk7 showing gene expression (box 1) and stages of oocyte development (box 2). Gene expression (box 1) includes biological processes and target genes (italic), which were enriched at specific time points during the experimental period. Identification of male signal is based on gene expression patterns in Wk5 females. Oocyte development stages are based on macroscopic observation and differential gene expression patterns associated with stage of oogenesis. Nomenclature follows stages based on histological studies of calanoid copepods [reviewed in]. Right: final stages of oocyte development (OS4), which include insemination and egg release. Wk 7.5 corresponds to the presence of the first clutch of egg releases by incubated females in the laboratory.