Larval growth and mortality
By day 4 post-fertilization, geoduck larvae had an average shell size of 131 µm. This increased to 146 µm on day 6 and 161 µm on day 8 post-fertilization. At day 4 post-fertilization, 3.1 million larvae were stocked in each tank. By day 10 post-fertilization there were 516,000 larvae in one tank and 1.3 million in the other. Ciliates were observed in the larval samples by day 8 post-fertilization; by day 10, there were high numbers of ciliates in the larval culture accompanied by high larval mortality.
Proteomics of larval development
The abundance of proteins in the geoduck larval proteomes (N = 6,133 proteins; Supplementary Table S1) are significantly distinct from each other across days 4 through 10 post-fertilization (ANOSIM R = 0.8889, p-value = 0.005; Fig. 1). Four proteins are strongly, positively weighted on NMDS1 axis and thus contribute significantly to proteomic changes over time – elongation factor 1 a (geoduck protein ID in Supplementary Table S1 DN61152_c1_g2_i1::g.31740), deleted in malignant brain tumors 1 protein (DN55033_c0_g1_i1::g.9300), peptidyl-prolyl cis-trans isomerase (DN40375_c0_g1_i1::g.39279), and tubulin alpha chain (DN61033_c0_g2_i3::g.6327).
Nonmetric multidimensional scaling plot of larval proteomes (N = 6,133 proteins) across days 4 through 10 post-fertilization. The points are lightest on day 4 and darkest on day 10. The two points representing proteomes on day 8 are directly on top of each other.
The strongest signal in the differentially abundant enrichment analysis comes from the changing abundance of proteins involved in translation over time. Many of these proteins increase in abundance over time. Between days 6 and 8 post-fertilization, proteins at elevated abundance on day 8 (n = 38 proteins) contribute to the enrichment of GO terms translational elongation (biological process, BP), and translational elongation factor activity (molecular function, MF). Between days 8 and 10, proteins at relatively higher abundance on day 10 (n = 138 proteins) are responsible for the enriched terms translation (BP), translational elongation (BP), ribosome (cellular component, CC), cytosolic large ribosomal subunit (CC), small ribosomal subunit (CC), structural constituent of ribosome (MF), and rRNA binding (MF).
Proteomic profile of larval development
Proteins regulating the progress of larval development were identified through comparison with similar proteomes from larvae raised at lower pH that did not experience ciliate-induced mortality. Functional categories identified in both datasets were determined to be involved in development, as opposed to the stress response. Diverse protein groups made up the full suite of differentially abundant proteins across developmental time. Proteins at elevated abundance on day 6 compared to day 8 (n = 28 proteins) included those involved in muscle activity, cytoskeletal structure, amino acid degradation, and carbohydrate metabolism. Proteins involved in muscle activity were four isoforms of twitchin (DN4488_c0_g1_i1::g.37991, DN84882_c0_g1_i1::g.7474, DN37585_c0_g1_i1::g.30792, DN54513_c0_g1_i1::g.20073). Cytoskeletal structure proteins included an axonemal 84 kDa protein (DN52327_c0_g1_i1::g.16221), two isoforms of spectrin beta chain (DN49196_c0_g1_i1::g.12832, DN63615_c0_g1_i4::g.19798), and dystonin (DN64009_c1_g1_i4::g.9078). An enzyme involved in glycine degradation (glycine cleavage system P protein – DN58628_c5_g1_i2::g.33280) and lysine degradation (alpha-aminoadipic semialdehyde synthase – DN102693_c0_g1_i1::g.40254) were elevated on day 6. The up-regulation of carbohydrate metabolism was supported by the inclusion of malic enzyme (DN55577_c7_g1_i1::g.30103), endoglucanase (DN59810_c3_g1_i1::g.27515), and transketolase (DN62420_c0_g1_i1::g.26365).
On day 8, proteins at increased abundance were involved in processes such as embryonic development, translation, stress response, and the cell cycle. Mesenchyme-specific cell surface glycoprotein (compared to day 6, DN60034_c1_g1_i1::g.37906), agrin (compared to days 6 and 10, DN50844_c0_g1_i1::g.3048), and delta-like protein (compared to day 10, DN64019_c1_g1_i2::g.9096) were at elevated abundance on day 8 and are responsible for tissue development and neuromuscular junctions during embryogenesis. Several proteins involved in protein translation, transport, and degradation were at higher levels on day 8, including ribosomal proteins (e.g., DN55459_c0_g2_i2::g.4318), elongation factors (e.g., DN61142_c1_g2_i1::g.31740), nucleolar protein 56 (DN57041_c0_g2_i1::g.3336), RNA polymerase-II associated factor (DN57191_c0_g1_i2::g.28950), and cathepsins (e.g., DN54782_c0_g1_i1::g.5290). On day 8 there may have been an up-regulation of the cell cycle with evidence from increased abundance (compared to day 10) of proteins involved in cell cycle control and the cytoskeleton (e.g., centrosomal protein of 78 kDa – DN44281_c0_g1_i2::g.1342, nuclear protein MDM1 – DN64001_c0_g1_i1::g.14401, antigen KI-67 – DN63195_c0_g2_i2::g.25524, suppressor of G2 allele of SKP1-like protein – DN63050_c5_g2_i1::g.279). Apoptosis proteins were at elevated abundance, including cell division cycle and apoptosis regulator protein 1 (compared to day 10, DN55421_C0_g1_i1::g.4308) and programmed cell death protein 4 (compared to days 6 and 10, DN60246_c2_g3_i2::g.7173). Cilia biogenesis was also important at this time point with an increased abundance of proteins such as centrin-4 (DN30560_c0_g1_i2::g.39913) and dynein heavy chain 7, axonemal (DN52275_c0_g1_i1::g.16099), cilia- and flagella-associated protein 58 (DN63766_c0_g1_i4::g.4722), and intraflagellar transport protein 57-like (DN62855_c0_g2_i1::g.20768). Four of the proteins that were at elevated abundance on day 8 were glycoproteins: mesenchyme-specific cell surface glycoprotein (DN60034_c1_g1_i1::g.37906), prosaposin (DN63880_c2_g1_i3::g.30762), agrin (DN50844_c0_g1_i1::g.3048), and fibronectin type-III domain-containing protein (DN55720_c0_g1_i1::g.14093).
The most significant proteomic signal on day 10 was a surge in abundance of ribosomal proteins with other proteins at higher abundance in categories such as protein folding, tissue growth/restructuring, and energy metabolism. Dozens of ribosomal proteins, several elongation factors, and other proteins associated with translation were at increased abundance on day 10. As a likely balance to the surge of new proteins, several proteins involved in protein degradation (aminopeptidase N – DN60972_c0_g2_i1::g.12028, cysteine peptidase – DN24514_c0_g1_i1::g.3095) and protein folding (a suite of heat shock proteins – e.g., DN56353_c0_g1_i1::g.29541, peptidyl-prolyl cis-trans isomerase – DN40375_c0_g1_i1::g.39279, calreticulin – DN42620_c0_g1_i1::g.2821, and glucose-regulated protein 78 – DN41738_c0_g1_i2::g.3144) were also detected at elevated levels on day 10. There is also evidence of tissue restructuring with an increase in abundance of proteins involved in muscle growth (kyphoscoliosis peptidase – DN54342_c0_g1_i1::g.35621, actin – DN40952_c0_g1_i1::g.3693, twitchin – DN84882_c0_g1_i1::g.7474), vascularization (basement membrane-specific heparan sulfate proteoglycan core protein – DN1574_c0_g1_i1::g.30025), and embryogenesis (multiple epidermal growth factor-like domains protein – DN61224_c0_g1_i1::g31991). There was also an increase in abundance of proteins involved in metabolism and energy production: acetyl-CoA C-acyltransferase, succinate-CoA ligase [ADP/GDP-forming] subunit alpha, ATP/ADP translocase, and adenosine kinase (DN65641_c0_g1_i1::g.38953, DN5111_c0_g1_i1::g.3560, DN31265_c0_g1_i1::g.39364, DN86572_c0_g1_i1::g.31553).
Proteomic profile of larval immune response
There were 855 proteins identified in the pH 8.2 dataset that were not detected in larvae reared in parallel at lower pH where there was no ciliate presence. Some of these proteins would have been detected in the current dataset because of their role in ciliate response; others may have been unique to this dataset simply due to the difference in ambient pH of the larval culture. In the enrichment analysis, compared to all proteins in the pH 8.2 dataset, enriched terms included integral component of membrane (CC), membrane (CC), regulation of transcription DNA-templated (BP), and DNA-binding transcription factor activity (MF). Eleven of these proteins were significantly differentially abundant (Fig. 2), 10 of which were highest at day 10 and most of which are ribosomal proteins.
Proteins hypothesized to be involved in the larval stress response to ciliate infestation. The clustered protein group dendrogram (panel A) shows the 4 clusters of proteins in red boxes with the cluster number at the bottom of each box. Panel B shows the abundance profile for each protein across day 4–10 post-fertilization with abundance plots grouped by cluster (cluster number is in the header of each plot). Annotations for the proteins in each plot are listed within each plot. All proteins are likely geoduck larval proteins except for cruzipain (cluster 1), which is a parasitic virulence protein.
On day 8, ciliates were first observed in the larval culture. In addition to the proteins described in the previous section, stress response proteins also increased on day 8 compared to days 6 and 10, including two isoforms of heat shock protein 70 (DN56353_c0_g1_i1::g.29541, DN56353_c0_g1_i2::g.29542), a thaumatin-like protein (DN55757_c0_g1_i1::g.14062), glutamate-cysteine ligase catalytic subunit (DN62819_c0_g1_i1::g.20739), glutathione peroxidase (DN60231_c1_g1_i1::g.7155), glutathione reductase (DN61098_c8_g3_i1::g.6407), and superoxide dismutase (DN61210_c0_g1_i3::g.32050) (Fig. 2).
The ciliate infestation had increased by day 10, along with larval mortality (see above). The parasite virulence factor cruzipain (DN76433_c0_g1_i1::g.22190) was first detected on day 8 in the proteomics data and was at significantly elevated abundance by day 10 (Fig. 2).
Source: Ecology - nature.com
