PC1/3 has a pre-bilaterian origin and is conserved in all insect groups except flies
To understand the evolutionary history of the insect subtilisin-like protein convertases and to map patterns of gene loss, we scored genomes of selected species for presence or absence of these enzymes. There are seven PC-proteins in mice and humans (PC1/3, PC2, PC5, PC4, PC6/PACE4, PC7 and furin), in addition to PCSK-9 and SKI-1, which also contain subtilisin-like catalytic domains but are membrane bound . We surveyed well-assembled genomes representing major insect lineages in addition to other animal lineages. We then used identified PC protein sequences for gene family reconstruction (see gene tree Fig. 1A), using SKI-1 proteins as an outgroup (PCSK-9 was not found in any invertebrate genome).
When comparing different bilaterians it became evident that a set of four PCs is shared between disparate groups: PC1/3, PC2, PC7 and furin (see Fig. 1A, B). These were found in representatives of arthropods, molluscs and vertebrates, representing the major bilaterian lineages. Nematostella vectensis, belonging to the basally branching radial symmetric cnidarians, has a clear PC1/3 and a PC7 orthologue, and one gene that groups with furins, but lacks PC2. In addition, there are six cnidarian PCs that do not group with any of the bilaterian convertases but form a group of their own (Fig. 1A). The vertebrate furin proteins have undergone an expansion. Vertebrate PC4 and furin are closely related and both cluster with invertebrate furin1. The PC5 and PACE4 proteins cluster with invertebrate furin2 and are closely related to one another (Fig. 1A).
When compared to vertebrates, the PC protein family is more compact in insects, typically comprising PC1/3, PC2, furin1 and furin2 (with some additional lineage specific duplications of furin genes in hemipterans, lepidopterans and collembolans Fig. 1A, B). Two furin genes are the typical gene content in invertebrates as direct orthologues to the insect furins are present in the other invertebrate taxa such as chelicerates, myriapods and the lophotrochozoan Lottia gigantea. In the myriapod Strigamia maritima, an arthropod with a conservative genome organisation  a PC7 gene is present. It was lost, presumably independently, from spiders and all hexapod genomes. By contrast PC1/3 is conserved in all insect groups, but no orthologues were detected in the dipteran genomes that we sampled (Drosophila melanogaster and Aedes aegypti). In addition, BLAST searches for PC1/3 genes in the entire dipteran clade did not identify any orthologous sequence (last verified in 05–2021). Hence the factor was specifically lost in the lineage leading to the Diptera. Similar to flies, Caenorhabditis elegans, the other major invertebrate model organism, has lost its PC1/3 orthologue but only possesses a clear PC2 orthologue .
To score the degree of conservation of the Tribolium prohormone convertases, we compared the protein sequences. On the protein level the Tribolium neuroendocrine specific convertases PC1/3 and PC2 share moderate overall sequence similarity of 44%. However, they have a near identical organisation. An N-terminal signal peptide that directs the polypeptides to the neurosecretory pathway is followed by an S8-pro- and S8-peptidase catalytic domain exerting the enzymatic functions, and a P/homo B regulatory domain (see Fig. 1C). This domain structure is conserved between the Tribolium and vertebrate neuroendocrine specific PCs . A specific amino acid motif with a core of -RGD- has been identified within the active domain of vertebrate PC1/3 and PC2 and was shown to be required for sorting the vertebrate enzymes into the secretory vesicles . This motif is present in Tribolium PC2 and in all other insect PC2 proteins that we have looked at. However, the motif is absent from the PC1/3 protein of Tribolium and other insects, and from the myriapod Strigamia, but present in the cnidarian and chelicerate PC1/3 proteins.
PC2 is expressed ubiquitously in cells of the nervous system, whereas PC1/3 is restricted to identifiable cell populations
PC1/3 has not been studied in any insect/arthropod before. Given that Tribolium contains the arthropod-typical set of two PCs, we asked, whether PC1/3 and PC2 were expressed in different or overlapping cell groups. We performed RNA in situ hybridisation to detect specific expression of these genes in embryos and in the larval nervous system of Tribolium. Limited sequence similarity between the paralogues on DNA-level allowed for the production of specific RNA-probes (see Additional file 1: Figure S1).
We found that PC2 is expressed during late embryogenesis (NS 14, which is still a flat germband with the tips of the legs reaching the anterior part of the second posteriorly following segment ) in many cells of the head and trunk neuroectoderm (Fig. 2A). At a slightly later stage (NS 15, shortly before hatching when the whole embryo is of oval shape ) most neural cells of the developing central nervous system express PC2 (Fig. 2B). At the larval stage, PC2 expression is found in most cells of the nervous system (Fig. 2C). A more intense expression is seen in the anterior medial brain area in larvae (arrow in Fig. 2C) where the pars intercerebralis is located, a structure of the insect brain with known neuropeptidergic properties .
By contrast, we did not find any embryonic expression of PC1/3 (see Additional file 1: Figure S2). Strikingly, expression in the larval nervous system was restricted to individual cells. In the suboesophageal ganglion we found two dorsally located cell groups. The anterior cluster comprised two cells (white arrow in Fig. 2D) and the more posterior normally contained 6 cells (white arrow in Fig. 2D). In one of four inspected nervous systems the posterior cluster comprised 8 cells, indicating some variability. Expression was also found laterally in the posterior brain (tritocerebrum) where two cells on each side stain positive (arrowheads Fig. 2D). We found no signal in the PI or other cells of the protocerebrum, nor in the mid-thoracic ganglia. With the methods available to us, we were not able to screen for PC1/3 or PC2 expression in the peripheral nervous system or neuroendocrine glands.
Divergent functions of PC1/3 and PC2 in larval growth
Given that most arthropods have both PC1/3 and PC2 and given the different expression patterns in Tribolium we hypothesised that these enzymes would have important and at least partially different functions in larvae. To this end we used larval RNAi to test for effects of PC1/3 and PC2 gene knockdown on larval development. The use of two non-overlapping fragments produced similar effects, indicating that the phenotypes were specific (also see Additional file 1: Figure S1 for a DNA sequence alignment and annotation of the dsRNA fragments, and see Additional file 1: Figure S3 for quantification of knockdown by qPCR).
We first injected dsRNA targeting the respective genes into 12-day-old L4 larvae. At 32 °C Tribolium undergoes 6 larval moults (L1–L7) and larval stage 4 normally starts from day 11 after egg lay, although there is some natural variation within this timing. On day 12 80% of larvae had completed the 3rd larval moult to L4 and no larvae were at stage L5 by then. We hand-picked average-sized larvae to exclude differing stages from the experiments.
We also included a double knockdown of both factors in our analysis. Growth curves and moult cycles were recorded following the injections.
Wild-type and dsRed dsRNA-injected control animals showed a constant weight gain until they reached a weight of over 2 mg (Fig. 3A and see Additional file 1: Figure S4 for growth curves of untreated and dsRed dsRNA control-injected larvae). Then the larvae lost some weight as they stopped feeding in preparation for the larva to pupa transition (Fig. 3A, S4A). By contrast, PC1/3 RNAi-treated larvae stopped growth almost completely for several days (Fig. 3B). Some of the observed larvae showed near zero weight gain over a period of up to 30 days, and some of these individuals died prematurely (larval lethality was 50%). Another half of the larvae did initially show a flat growth curve but entered a phase of increased weight gain later, presumably when the RNAi-effect had faded out over time, and eventually reached a weight sufficient for larva to pupa transition. Due to the slowed growth the larval period was significantly prolonged in PC1/3 knockdown larvae: all control larvae had pupated by day 28 of development whereas 50% of PC1/3-RNAi specimens remained in the larval period beyond the age of 41 days. In an independent experiment, we observed an individual PC1/3-dsRNA-treated animal that had not pupated even after 3 months’ time. Intriguingly, we observed supernumerary moults in the knockdown larvae (see below).
PC2 dsRNA-injected larvae had a high mortality and half of them had already died on day one after injection (Fig. 3C), compared to zero deaths in control-injected larvae at this timepoint. Unlike PC1/3 knockdown larvae PC2-RNAi larvae initially showed some weight gain, but still lagged behind the control sets (see Fig. 3C, E). All PC2-RNAi larvae eventually suffered rapid weight loss and were found dead shortly after. Larvae that were co-injected with PC1/3- and PC2-dsRNA, each at the same concentration of 1 µg/µl dsRNA targeting each gene, showed a strongly reduced growth as known from the PC1/3 knockdown and a high lethality as observed in PC2 larvae (Fig. 3D) indicating additive function. Results of independent repeat experiments confirming the effect of PC1/3 and PC2 knockdown in larvae are shown in Additional file 1: Figure S5.
In conclusion, these results showed that PC1/3 and PC2 are both required to maintain larval growth and survival. The different dynamics, however, indicated that both function independently at the larval stage.
PC1/3 knockdown leads to the decoupling of growth from moulting
Under normal conditions wild-type Tribolium castaneum undergo 6 larval moults and the timing of larval moulting is thought to depend on the growth rate. We wondered how the reduced growth in PC1/3 and PC2 knockdown would affect the number of moults. Therefore, we plotted the moults on the average growth curves based on the above-described experiments (Fig. 3E, green circles indicate the point in time when most animals moulted, the green dotted line marks the time range when moults of the remaining larvae occurred). Most (5/8) PC2 injected larvae underwent one moult after some weight gain, but accompanied by the high lethality, the majority of these larvae (4/5) did not go through the second moult cycle. Only a small proportion of PC1/3 and PC2 double RNAi animals did complete one moult (3/8), and none of them completed a second moult (Fig. 3E). Unexpectedly, larvae treated with only PC1/3 dsRNA still went through larval moults despite strongly reduced growth (see above). They underwent the first two moults at similar points in time compared to control injections although there was almost no weight gain during that period (Fig. 3E). Some individuals went through moults without effective weight gain since the last moult (see Additional file 1: Table S4). The third moult was delayed by more than four days compared to the last larval-larval moult of the wild type. Interestingly, the PC1/3 knockdown larvae went through supernumerary moults while the increase in weight was minimal.
Taken together, our experiments showed that PC1/3-knockdown led to a strong inhibition of larval growth and to the decoupling of larval moults from the growth process.
Amontillado-like ecdysis phenotypes occur at a high penetrance in PC2 knockdown, and at a low frequency, in PC1/3 knockdown larvae
Mutants of the Drosophila PC2 orthologue amontillado show a failure of the moulting process: the larva produces a new cuticle but is not able to shed its old cuticle and becomes “locked-in”, which then leads to weight loss and death . Hence, Tribolium PC2-RNAi larvae were specifically screened for this phenotype once they had died. Indeed, a second cuticle was detected, unambiguously recognisable by the duplicated mandibles and terminal structures (Fig. 4A, A’).
By screening a number of dead PC2 knockdown larvae (injected at 12 days) we found, that 75% (n = 20) (see Table 1) of them suffered from an ecdysis phenotype and had a second cuticle like shown in Fig. 4. The remaining were very small when found dead, but no second cuticle was found. Somewhat surprisingly we also found this phenotype in PC1/3-RNAi larvae but at much lower penetrance. Survival of PC1/3 larvae was 50%, and 26% of dead larvae showed the ecdysis phenotype (n total = 38, n of inspected dead larvae = 19), which gives a total penetrance of the phenotype of 13% (see Table 1). The failure in the moulting process and the induction of supernumerary moults in PC1/3 RNAi indicate that the gene acts pleiotropically in multiple steps of moulting control. The results also indicate that both PC1/3 and PC2 act in the moult pathway and that the other protease may be able to partially rescue the process in the knockdown larvae. To test this, we screened larvae in which a double knockdown was performed (as shown in Fig. 3D) for an ecdysis phenotype. We found that 100% of these larvae (n = 20) suffered from this phenotype (see Table 1 for comparison), supporting the hypothesis of collaborative function in this process. In addition, we evaluated which proportion of the RNAi-larvae were able to successfully complete one or two moults (Table 1), before either dying of an ecdysis phenotype, or other causes, or surviving to the pupal stage (PC1/3 RNAi larvae only). We found that 65% of PC2-RNAi larvae completed at least one moult cycle, compared to only 33% of the double knockdown larvae. None of the latter was able to complete a second moult cycle, whereas 15% of the PC2 single knockdown were able to do so. All PC1/3 knockdown larvae completed one moult and the large majority (89%) also completed a second moult, reflecting the low penetrance of the phenotype in this knockdown. Taken together, PC1/3 and PC2 act together in the moult process and can partially compensate for one another’s function while the contribution of PC2 to this process is bigger.
Both PC2 and PC1/3 are essential for survival and fertility of adult beetles
To test the function of both genes at the adult stage we performed pupal RNAi. Female pupae where injected at a mature pupal stage: we picked pupae with pigmented eyes and sclerotised mandibles. In untreated animals eclosion occurs 1–2 days after the appearance of these features. Injections sometimes slightly delay the process. Therefore, deaths occurring up to 5 days after injection may partially be caused by injection injury (compare dsRed control-injected set in Fig. 5A).
We found that interfering with expression of PC2 and PC1/3, respectively, led to severe reduction of survival rates compared to control-injected animals (Fig. 5A). Over the experimental time course of 41 days PC2-RNAi beetles (n = 100) had a total survival rate of only 20% whereas 48% of PC1/3-RNAi beetles survived, compared to 80% survival in the control (injected with dsRed-dsRNA, n = 30). A repeat experiment confirmed the reduced viability of PC1/3- and PC2-dsRNA injected beetles when compared to a control set: after 30 days 44% of PC1/3-RNAi beetles survived, 38% of PC2-RNAi beetles and 67% of a control set (n = 100/each RNAi and n = 15/control; see Table 2 for total frequencies of adult phenotypes).
Beetles of both knockdowns that died in the first few days after injection (up to day 5, see Fig. 5A) typically showed signs of incomplete eclosion (Fig. 5B, C, Table 2): the wings were not fully covering the abdomen. Abdomens were thin and of a dry appearance. The desiccation is possibly a result of the incomplete covering with the elytra.
Also, later in the course of the experiment, beetles with fully elongated elytra showed signs of starvation and possibly desiccation (Fig. 5D, E, also see Table 2). Wings were frequently half opened when dead, but since these beetles had gone through complete metamorphosis before, we assume that problems with metabolism and water balance led to this appearance and to their death.
To test for effects on female fertility we mated females that were injected as pupae (n = 100 for each gene) with untreated male beetles (ratio 5:1) in a bulk mating. We assessed their reproductive success at different time points by counting all eggs in a 24-h period. We started from day 8 in order to include only adults that had successfully eclosed. We found that the number of eggs was severely reduced in both treatments. From more than 10 eggs/female/24 h egg laying decreased to less than 2 eggs/female/24 h in PC2 and PC1/3 knockdown animals alike (see Fig. 6A). The small number of laid eggs developed at a normal rate to wild-type appearance. An independent repetition of this experiment confirmed a lasting reduced fertility of less than 1 egg/female/24 h over 30 days, compared to 7–11 eggs/female/24 h in the control (n = 100/ each RNAi and n = 15/control, not shown).
We then tested involvement of PC1/3 and PC2 function in male fertility by injecting male pupae and crossing 30 individual males to wild type female beetles at a ratio of 1:5. Survival of males following injections was also reduced over the experimental time course of 14 days: PC1/3-RNAi: 36%, PC2-RNAi: 42%, dsRed-ctrl.: 86%. We did find significantly reduced fertility of PC1/3 RNAi males and even more severely in PC2 RNAi beetles (Fig. 6B). In line with a partial recovery of injected beetles from the RNAi-effect, the reduction of male fertility grew less severe the more time had passed since injection. In addition, even though egg number was reduced significantly at all three timepoints, females mated to injected males still laid a good number of eggs (Fig. 6B), and effects were generally milder than observed for female RNAi-beetles. The majority of these eggs were fertilised and developed into larvae at a rate not different from wild-type eggs (ctrl: 92% (n = 106), PC1/3-RNAi: 93% (n = 71), PC2-RNAi: 88% (n = 72)). These numbers indicate the presence of functioning sperm. Variation of egg number produced by individual matings was high and the death rate of the injected males was also high. The lowest number of eggs was usually obtained from males that would die shortly after. Together with the comparably moderate decrease of fertility, this suggests that the significant reduction of egg number following male treatment may have been a consequence of reduced overall fitness rather than a specific failure of sperm production or mating.
Ovarial phenotypes underly the female infertility of PC1/3- and PC2-RNAi beetles
We aimed to further understand the causes of female infertility in PC1/3- and PC2-RNAi beetles by analysing in how far the pupal knockdown affects the ovary structure and/or oocyte maturation. In Tribolium the development of the telotrophic meroistic ovaries starts at the larval stage when germ cells proliferate and form 5–6 distinguishable clusters on each side. Mitotic waves of germ cells continue at the pupal stage . Maturation of ovarioles and enclosure of the young oocytes with follicle cells takes place in the period from 0–5 days post adult eclosion [30, 31]. Typical number of ovarioles in adult Tribolium is five per side .
First, we identified three classes of ovarial phenotypes (Fig. 7A, B): normal/wild-type-like, a so-called held-egg phenotype in which mature eggs accumulate in the oviduct without being laid, and a ‘small-ovary’ phenotype in which ovarioles and hence the whole ovaries are significantly smaller than the wild-type ones. We found a comparable distribution of phenotypes in both knockdowns (Table 3). Most ovaries were of the ‘small’ appearance (PC1/3: 62%, PC2: 54%), a small percentage (10% in each knockdown) were classified as ‘held-egg’, while the remaining ovaries were wild-type like.
Even though we focused on the timepoint of 14–15 days after injection, inspection of a small number of ovaries at 5 and 30 days revealed similar phenotypes. We further visualised the structure of the ‘small-ovary’ phenotype by fluorescently labelling them for actin (using phalloidin) and for nuclei (using DAPI). When comparing the ‘small ovary’ phenotype to wild-type ovaries (Fig. 8A) we found that all components of an ovariole were still present in the ‘small ovary’ phenotype (Fig. 8B, C). Like wild type, they comprise a terminal germarium followed by egg chambers with oocytes of consecutive developmental stages (mature oocyte marked by white arrowheads in Fig. 8A). In mild phenotypes the shapes of the ovarioles were not altered and the oocytes filled the whole egg-chambers, with their membrane lining the outer follicle cell sheet (Fig. 8B) like in the wild type. However, the most mature oocytes of this mild phenotype did not reach the same size as they would in wild-type ovaries (white arrowheads in 8A and B) and the lateral oviducts (red asterisks in Fig. 8A–D) were not filled with eggs as in the wild type. This indicates that these ‘small ovary’ phenotypes were not capable of producing viable eggs. The more severe form of the ‘small ovary’ phenotype (Fig. 8C) showed more slender proportions of ovarioles caused by the oocytes having a strongly reduced diameter. The oviducts are also not filled with mature eggs (red asterisks, Fig. 8C). Such defects could be primary effects of impeded neurosecretory signals or secondary due to starving of the animal. In order to distinguish these possibilities, we analysed wild-type animals, that had been starved for 4–10 days directly after eclosion. Indeed, PC knockdown ovaries looked similar to ovaries in beetles that suffered starvation (Fig. 8D, starved for 10 days, we did not find notable differences of the appearance of ovaries starved for 4, 8, 10 or 12 days, respectively). Note that severe and less severe forms of the ‘small ovary’ phenotypes were seen in both, PC2 and PC1/3 knockdowns. In the ‘small ovaries’ of the severe form, as in the starved ovaries, the most mature oocytes remained small and did not have the typical oval shape. There were often gaps between the follicle cells and the oocyte membrane (compare wild-type oocyte in Fig. 8E and mild form of ‘small ovaries’-oocyte in Fig. 8F to Fig. 8G and H, red arrows). We also observed a disintegration of some oocytes (white arrows in Fig. 8C), which might be followed by reabsorption of the material by the follicle cells. No mature oocytes were located in the lateral oviducts of starved ovaries (red asterisks, Fig. 8D). Intriguingly, most of the severely reduced ovaries from both knockdowns had a slightly increased number of ovarioles: Tribolium ovaries normally comprise 10 ovarioles whereas the severely reduced ones often had 11–12 ovarioles (see for example Fig. 8C). We also occasionally found this in ovaries from starved beetles, but never in wild-type ovaries. Due to the small number of ovaries that were fully intact after dissection and staining procedures we were not able to quantify this effect and it is also not clear how pupal RNAi or starvation from adult eclosion onwards could have caused this, given the pre-formation of ovarioles at the larval stage .
In summary, we found that the dominant appearance in both PC1/3 and PC2 knockdown was the ‘small ovary’ phenotype in which oocytes failed to reach a mature size and form. This effect could be reproduced by depriving the beetles of food, indicating insufficient availability or distribution of nutrients to the oocytes in the RNAi-beetles. Alternatively, given the specific role of a number of neuropeptides in the process of yolk deposition to the oocyte  this may also be a specific effect of impaired neuropeptide signalling. Note that the different classes of ovarial phenotypes taken together do not occur at a frequency that would fully explain the reduction in egg number. A proportion of 28% of PC1/3- and 36% of PC2-RNAi ovaries were of wild-type appearance, whereas egg number was reduced to less than 20% in both knockdowns at 16 days. Therefore, we cannot exclude additional fitness-related or behavioural effects caused by interfering with the neuroendocrine system.