
==== Front
Curr Res Insect Sci
Curr Res Insect Sci
Current Research in Insect Science
2666-5158
Elsevier

S2666-5158(24)00024-6
10.1016/j.cris.2024.100094
100094
Research Article
A Rhodnius prolixus catalytically inactive Calpain protease patterns the insect embryonic dorsal-ventral axis
Julio Alison ab
Guedes-Silva Tainan C. a
Berni Mateus ac
Bisch Paulo Mascarello b
Araujo Helena haraujo@histo.ufrj.br
ac⁎
a Institute for Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
b Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
c Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil (INCT-EM)
⁎ Corresponding author at: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, CCS Bloco F – sala F2-031, Cidade Universitária – Ilha do Fundão, Rio de Janeiro, RJ, 21.941-902, Brazil. haraujo@histo.ufrj.br
17 8 2024
2024
17 8 2024
6 10009430 12 2023
14 8 2024
15 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• The hemiptera Rhodnius prolixus dispalys an expanded set of Calpain-encoding loci.

• Catalytically inactive Calpains arose several times in insect evolution.

• The catalytically inactive Rp-Calpain A/B2 controls embryonic dorsal-ventral patterning.

The calcium dependent Calpain proteases are modulatory enzymes with important roles in cell cycle control, development and immunity. In the fly model Drosophila melanogaster Calpain A cleaves Cactus/IkappaB and consequently modifies Toll signals during embryonic dorsal-ventral (DV) patterning. Here we explore the role of Calpains in the hemiptera Rhodnius prolixus, an intermediate germband insect where the Bone Morphogenetic Protein (BMP) instead of the Toll pathway plays a major role in DV patterning. Phylogenetic analysis of Calpains in species ranging from Isoptera to Diptera indicates an increase of Calpain sequences in the R. prolixus genome and other hemimetabolous species. One locus encoding each of the CalpC, CalpD and Calp7 families, and seven Calpain A/B loci are present in the R. prolixus genome. Several predicted R. prolixus Calpains display a unique architecture, such as loss of Calcium-binding EF-hand domains and loss of catalytic residues in the active site CysPc domain, yielding catalytically dead Calpains A/B. Knockdown for one of these inactive Calpains results in embryonic DV patterning defects, with expansion of ventral and lateral gene expression domains and consequent failure of germ band elongation. In conclusion, our results reveal that Calpains may exert a conserved function in insect DV patterning, despite the changing role of the Toll and BMP pathways in defining gene expression territories along the insect DV axis.

Keywords

Calpain
Dorsal-ventral patterning
BMPs
Embryonic development evo-devo
Rhodnius prolixus
Chagas disease
==== Body
pmc1 Introduction

Signaling pathways are deployed recurrently in several developmental and physiological contexts. To perform this multitude of functions, signaling networks should account for the incorporation of novel elements and changes in their epistatic relations. The Toll and BMP signal transduction pathways are deployed for embryonic dorsal-ventral (DV) patterning in several animal species, setting up gene expression territories that will give rise to the mesoderm, neuroectoderm and extraembryonic tissues. In vertebrates such as Mus musculus and Xenopus laevis the BMP pathway sets up the DV axis, while Toll signals are mostly dedicated to innate immunity (Bier and De Robertis, 2015). In insects, the importance of each pathway in DV patterning varies among the different insect orders (Berni et al., 2014, 2023; Sachs et al., 2015; Pechmann et al., 2021). For instance, in the fly D. melanogaster and the coleoptera T. castaneum, the Toll pathway subdivides the entire DV axis, while the BMP pathway exerts a limited role to subdivide the dorsal ectoderm and extraembryonic tissues (Nunes da Fonseca 2008; Reeves and Statopoulos 2009). Conversely, BMPs exert a major role to pattern the DV axis in the hemiptera O. faciatus and R. prolixus (Sachs et al., 2015; Berni et al., 2023). Based on this changing role, it has been suggested that BMPs display an ancestral role to pattern the DV axis, while the Toll pathway was coopted from its function in innate immunity to perform a role in insect embryonic development (Lynch and Roth 2011; Roth, S., 2023).

Calcium-dependent cystein proteases of the Calpain family modify Toll signals in the vertebrate (Han et al., 1999; Shumway et al.,1999; Scholzke et al., 2003; Chen et al., 2007; Wu et al., 2014) and invertebrate (Fontenele et al., 2013; Araujo et al., 2018) immune system. Classical Calpains target IκB homologs for cleavage, which decreases or modifies their ability to interact with NFκB superfamily transcription factors, resulting in changes in target gene expression (Shumway et al.,1999; Schaecher et al., 2004; Fenouille et al., 2012; Li et al., 2014). During Drosophila embryogenesis Calpain A (CalpA) cleaves Cactus/IκB and modifies signals from the Toll pathway (Fontenele et al., 2013). Therefore, Calpains seems to perform an evolutionarily conserved role to modify Toll signals. Based on the hypothesis that the Toll pathway was coopted from an immunity related role to patterning of the insect DV axis (Lynch and Roth 2011; Roth et al., 2023), it is reasonable to suggest that Calpains may also have been coopted for a Toll related function during embryonic patterning. To explore this possibility and try to understand how this signal transduction network evolved, it would be interesting to characterize the function of Calpains and their relations to the Toll and BMP pathways in an insect species where the role of Toll in embryonic DV patterning is limited. In that sense, we have analyzed the Calpain system of the triatomine R. prolixus, carrier of the protozoan Typanosoma cruzi and thus vector of Chagas disease.

The Calpain proteases are cytosolic, calcium-dependent intracellular cysteine protease that cleave a specific set of protein substrates at a small number of sites (Croall and Ersfeld, 2007; Sorimachi et al., 2011; Araujo et al., 2018). The modulatory activity of Calpains produces new protein fragments from substrate cleavage, which may develop new protein functions (neoproteins). Therefore, the neoprotein is a functional cleaved protein with roles distinct from the original molecule (Lopez-Otin and Overall, 2002). The general structure of a classical Calpain comprises four major domains: PC1 and PC2, that form the catalytic domain termed Calpain-type Cysteine Protease conserved (CysPc) module or Protease core, the membrane anchoring Calpain-type Beta-Sandwich domain (CBSW, previously known as domain III), and penta-EF-hand (PEF) calcium binding domain (Campbell and Davies, 2012; Araujo et al., 2018). Some Calpain homologues show substitutions in one or more of the well-conserved active-site triad residues, resulting in proteins devoid of enzymatic activity. These include the human CAPN6, several schistosome and nematode Calpains, and Drosophila Calpain C (Sorimachi et al., 2011). Different from vertebrates, classical invertebrate Calpains are monomeric.

Calpains are divided in classical and non-classical groups. Members of the non-classical Calpain cluster show a high degree of divergence from the classical Calpains (Ono and Sorimachi, 2012). Eukaryotic representatives of this family present a conserved CysPc domain, and most contain a CBSW domain. However, additional domains include Zn-finger and RanBP2 type motifs, such as those found in Calpain D encoded by D. melanogaster small optic lobes (sol; Delaney et al., 1991), or a transmembrane domain such as that found in Arabidopsis thaliana DEK1 (Lid et al., 2005).

Here we identify the complete set of R. prolixus Calpains, comprising one representative of each of the non-classical Calpains, as well as seven loci that encode classical Calpains. Based on functional analysis, our results suggest a function for CalpA/B1, CalpA/B4 and CalpC in oogenesis and we identify Rp-CalpainA/B2 as having a function during embryonic DV patterning, despite the lack of active site residues.

2 Methods

2.1 Insect rearing

R. prolixus rearing was performed at 28 °C (± 1 °C) and 70–75 % humidity, with animals fed on rabbit blood throughout all five nymph stages until reaching adulthood at approximately 5–6 months. Animal care and experimental protocols were conducted following the protocol approved by the Ethics Committee on Animal Use at UFRJ, under registration 123/22. Technicians dedicated to the animal facility at the Institute of Medical Biochemistry (UFRJ) conducted all aspects related to rabbit husbandry under strict guidelines to ensure careful and consistent animal handling.

2.2 Gene identification and phylogenetic analysis

We identified Calpain-like proteins by BLASTp using insect proteomes available at the Universal Protein Research (UniProt) (UniProt Consortium, 2007) and tBLASTn in VectorBase using D. melanogaster Calpain A, Calpain B, Calpain C and Calpain D and human Calp7 query amino acid sequences in our initial search. Evolutionary history was inferred from amino acid sequences, in accordance with Hall B.G., 2013, using the Maximum Likelihood method. The analysis included a total of 9 species and 60 complete amino acid sequences, which were aligned using multiple sequence alignment with high accuracy and high throughput (MUSCLE) (Edgar, 2004) with standard features. The evolutionary histories were inferred by Molecular Evolutionary Genetics Analysis version (MEGAX) (Kumar et al., 2018), and visualized using Interactive Tree of Life v2 (iTOL) (Letunic and Bork, 2021). The trees were validated by 500 bootstraps replications. The protein IDs are indicated in the trees.

For the analysis of CalpainA/B restricted to the CysPc domain, we used a highly conserved region of approximately 300 amino acids within the catalytic domain. CalpA of D. melanogaster was used as query, containing the entire CysPC domain, based on ProSite (ID: PRU00239).

2.3 Knockdown assays

Functional analysis by parental RNA interference (pRNAi) followed Berni et al., 2014. Double strand RNA (dsRNA) was synthesized from DNA fragments generated by 2 rounds of PCR. For the first round, primers containing sequences to amplify specific Calpain genes plus short (8 nucleotides) overhangs for recognition by the T7 Universal forward and reverse primers (Supp. Table S1) using cDNA from embryos or ovary as PCR template. For the second PCR, 2ul of the first reaction was used as template for T7 universal forward and reverse primer amplification. For dsRNA synthesis, in vitro transcription was performed using the MEGAscript kit (Ambion), per manufacturer's instructions. 2 ul of each dsRNA (2 ug/ul) were injected in the abdomen of adult females, 5 days prior blood feeding. For each pRNAi assay, a parallel control was performed using dsRNA for GFP (Green fluorescent protein) or Mal (E. coli maltose binding protein gene; dsMal, Gene ID: 948,538).

2.4 Fecundity and fertility assays

Adult female injections were performed with a sterile 10 μl GASTIGHT#1701 Hamilton syringe, into the ventral abdomen, between the third thoracic and first abdominal segment, with 1 – 2μl of 2μg/μl dsRNA in DNAse/RNAse free water. The insects were blood fed 5 days after dsRNA injection, and after one week egg collection was initiated. Fecundity was determined in groups of ten females. Eggs were collected and counted every 48 h to screen for oviposition phenotypes (fecundity). Fecundity was thus defined as the number of eggs laid per day, divided by the number of females in the group. The hatch rate was used to screen embryo viability phenotypes (fertility), defined 20 days after egg lay at 28 °C, taking into consideration that wild-type embryogenesis lasts 14–15 days at this temperature. Hatch rate is thus defined as the percentage of eggs that hatch in relation to the total number of eggs laid over 7 days.

2.5 Embryo collection and in situ hybridization

Synchronized eggs were briefly washed with distilled water to remove debris and transferred to a 2 ml microtube with 1 ml of distilled water. The eggs were boiled for 90 s, the water was replaced by 1 ml of formaldehyde 12 % (in phosphate buffered saline, PBS) and fixed for 2 h (6–8 °C). After this period, the embryos were incubated with 1 ml of formaldehyde 4 % containing 0,1 % of Tween 20 (PBST) under agitation for at least 1 hour. Subsequently, the eggs were washed three times in PBST. After manual dechorionation with a fine forceps, embryos used for in situ hybridization, followed by DAPI nuclear staining (1 µg/µl). in situ hybridization was performed as in Berni et al., 2023.

2.6 Total RNA extraction, cDNA synthesis and RT-qPCR assays

For cDNA generation, total RNA was extracted from stage 5 embryos (24–30 h) as in Berni et al., 2014 or from ovaries dissected 7 days after blood feeding, containing trophoblastic, pre-vitellogenic and choriogenic stages using Trizol Reagent (Invitrogen) as per manufacturer instructions. Total RNA was treated with RNAse free Turbo DNAse (Ambion, Life Technologies) to remove genomic DNA traces. cDNA was synthesized from 1ug total RNA using in vitro High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative Real Time PCR (RT-qPCR) was performed on a QuantStudio 3 Real Time PCR system™ (Applied Biosystems) using PowerTrack SYBR Green Master Mix™ (Applied Biosystems). The relative gene expression was calculated using the comparative ΔΔCT method (Livak and Schmittgen, 2001), using Elongation factor 1 (Elf1) as reference gene as in Berni et al., 2014. Oligonucleotides used for RT-qPCR Elf1 and Rp-Calpains were used in the final concentration of 150 nM. The oligonucleotides used in RT-qPCR assays are listed in Table S1. All assays were conducted with biological triplicates and three to four technical replicates.

2.7 Statistical analysis

The normality of fertility and fecundity data was checked with Shapiro-Wilk (α−0.05) and visualized with a Q-Q plot. The homogeneity of the data was verified by Levene's test (α−0.05), and the search for outliers was conducted using box plots. The significance of R. prolixus functional assays was defined using the Mann-Whitney test. The results are shown as the median of independent experiments.

For RT-qPCR statistical analysis, Mann-Whitney non-parametric T-test (considering unpaired groups) was used from each experimental group in relation to the control group.

All graphs and statistical analyses were performed using GraphPad 8 software (GraphPad Software, San Diego, CA, USA). The level of significance is shown in each figure (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05). Detailed quantification data is shown in Supplementary material.

3 Results

3.1 Identification of R. prolixus Calpains

The R. prolixus genome harbors several peptidase encoding sequences, including calpain-like proteases (Henriques et al., 2017). Here, we set to identify the different types of Calpain-like proteins in R. prolixus and define their evolutionary relationship to insect Calpains. To that end, we performed de novo searches with D. melanogaster proteins CalpA, CalpB, CalpC and CalpD as query amino acid sequences against the insect Proteome database available at Uniprot. Since D. melanogaster CalpA and CalpB show similar domain architecture, with 54 % identity over the entire amino acid sequence (ClustalO), there was no clear distinction between the Calpain A/B like sequences herein displayed. From this analysis we identified seven classical R. prolixus Calpains A/B, one Calpain C, and one Calpain D (Fig. 1). In addition, one R. prolixus Calp 7 ortholog is identified in Vectorbase based on homology to vertebrate calpains.Fig. 1 Identification of classical and non-classical Calpains of R. prolixus. (A) Number of Rp-calpains compared to selected insects. Displayed are Calpain-like genes in representative species since the Ordovician (500mya). Hemimetabolous species: Zootermopsis nevadensis, (Isoptera), Rhodnius prolixus (Hemiptera), Cimex lenticularis (Hemiptera); and holometabolous species: Nasonia vitripennis (Hymenoptera), Tribolium castaneum (Coleoptera), Bombyx mori (Lepidoptera), Anopheles gambiae (Diptera), Drosophila melanogaster (Diptera). Daphnia pulex (Crustacean) was used as an outgroup. (B) Phylogenetic tree and domain architecture of Rhodnius prolixus Calpains. Full set of classical (A/B and C) and non-classical (D and 7) Rp-Calpains. Only sequences larger than 500 amino acid residues were used for tree building, based on the R. prolixus genome GCA_000181055.3, RproC3.5 and transcriptomic data.

Fig 1

For phylogenetic analyses we gathered sequences from at least one species per order and used Calpain loci from the Crustacean Daphinia pulex as outgroup (Fig. 1A). With respect to classical Calpains, phylogenetic analysis places the R. prolixus A/B Calpains inside two distinct groups (Fig. 2). One (Rp-CalpA/B3) shows highest similarity to D. melanogaster Calpain A and Calpain B. At least one representative of each species analyzed shares this characteristic, comprising proteins with the complete CysPc/CBSW/EF-Hand architecture, including conservation of the active site Cys-His-Asn aminoacid residues in CysPc. The only exception is T. castaneum CalpA/B KYB28414.1, which has no EF-Hand (Fig. 2). Among these, Calpains A/B from B. mori and T. castaneum show the largest N-terminal disordered regions, which is known from biochemical studies to harbor target sequences for Calpain autocleavage (Guharoy et al., 2016).Fig. 2 Insect Calpain A/B phylogenetic and structural analysis. A) Phylogeny of insect Calpains A/B. Confirmed or predicted proteins were identified from the species: Drosophila melanogaster (Dm), Anopheles gambiae (Ag), Tribolium castaneum (Tc), Nasonia vitripennis (Nv), Bombyx mori (Bm), Rhodnius prolixus (Rp), Cimex lenticularius (Cl), and Zootermopsis nevadensis (Zn). The crustacean Daphnia pulex (Dp) was used as outgroup. Numbers inside the tree represent the bootstrap support. In bold the coding sequences identified for R. prolixus. Rp-cluster gathers exclusively R. prolixus sequences. In red are the sequences with mutated catalytic residues, likely giving rise to catalytically inactive Calpains. B) Domain architecture of Calpains A/B with indicated domains and active site residues conserved (red) or mutated (yellow). R. prolixus CalpA/B are highlighted.

Fig 2

Other Calpains display greater divergence to D. melanogaster CalpA and CalpB. Interestingly, one cluster (Rp cluster) harbors exclusively R. prolixus Calpains, indicating that these paralogs arose from a series of clade specific duplication events that led to the expansion of R. prolixus A/B Calpains. Importantly, we identified two CalpA/B containing mutations at the CysPc active site: in Rp-CalpA/B 4 (RPRC013347) only His is conserved at the active site and in Rp-CalpA/B 2 (RPRC013350) all three active site residues as well as the EF-Hand are absent. Pfam (Mistry et al., 2021) prediction indicated that the CysPc from Rp-CalpA/B 2 and Rp-CalpA/B 4 are sequence-based catalytically inactive Calpains (hereafter termed dead-Calpains, for any Calpains containing mutations at CysPc active site residues), which is supported by previous biochemical data gathered from Calpains with active site (e.g. Cys>Ser) substitutions (Pal et al., 2003; Hanna et al., 2008). Alignment of R. prolixus “dead” Calpains A/B (Fig. S1) against Drosophila Calpain A shows that residues surrounding the active site are conserved, despite the active site substitutions. This indicates that the overall structure of the inactive protein is maintained, and possibly the interactions with putative substrates and interacting domains in the cell are preserved as well.

Close examination of active site residues in all the proteins herein analyzed reveals that two clusters comprised entirely of Calpains A/B with modifications in active site residues. We defined as catalytically inactive Calpains those with at least one substitution in an amino acid residue of the active site, resulting likely in catalytically inactive enzymes. From here on we refer to these as "dead" Calpains. In addition, these proteins contain no EF-Hand domains. In fact, two proteins harbor, instead, a MITb (microtubule-interacting and trafficking) domain. Hence, catalytically inactive Calpains A/B show the highest phylogenetic and domain architecture divergence among the Calpain A/B tree. The presence of catalytically inactive proteins in different clusters suggests that they may have originated several times in insect evolution. To best understand how catalytically inactive Calpains A/B appeared in the insect phylogeny we also performed phylogenetic analysis restricted to the CysPc domain. (Fig. S2). The resulting tree is very similar to the tree comprising the entire protein sequences (Fig. 2), indicating that catalytically inactive Calpains A/B arose independently at least three times in insect evolution. Based on the prevalence of Calpains A/B harboring loss of active site residues in the CysPc domain in several species analyzed, it is tempting to suggest that catalytically inactive Calpains arose independently by convergent evolution and perform fundamental functions in the insect order.

In contrast to Rp-Calpains A/B, phylogenetic analysis indicated low divergence within orthologous Calpain classes C (Fig. 3A), D (Fig. 3B) and 7 (Fig. 3C). The high conservation we found for Rp-Calpain C, Rp-Calpain D and Rp-Calpain 7 may be the result of an absence of duplication events, hence providing selective pressure to preserve the single copy of these genes, maintaining their current function across insects. However, it is still not clear the roles of these single copy Calpain genes in insect development.Fig. 3 Unique R. prolixus Calpains. Phylogeny of insect Calpains C (A), D (B) and 7 (C). The species analyzed were the same as in Fig. 2.

Fig 3

3.2 Functional analysis of R. prolixus Calpains

To address the function of R. prolixus Calpains, we initially examined published transcriptomes for the presence of transcripts corresponding to the sequences of putative Rp-Calpain loci. The detection of Rp-Calpain transcripts would allow us to determine whether the Rp-Calpain loci identified correspond to active genes or to pseudogenes. Furthermore, transcripts identified in oogenesis and embryogenesis datasets would help define those loci that may have a developmental function, particularly during the early stages of embryonic patterning.

Examining expression during oogenesis we found that most R. prolixus CalpA/B loci are expressed, albeit some at low levels, during the previtellogenic stages (Table S2; Fig. S3). Only Rp-CalpA/B3 shows high expression during this stage. In fact, Rp-CalpA/B3 (KQ034117_1,155,762–1,172,636) was found while performing tBLASTn against the previtellogenic stage transcriptome (Brito et al., 2018), as this gene was not previously annotated in R. prolixus genome. As for non-classical Calpains, Rp-Calp7 and Rp-CalpD show the highest expression levels across all Calpains identified in the previtellogenic transcriptome, while Rp-CalpC has expression levels comparable to Rp-CalpainA/B4 (Fig. S3). In vitellogenic and choriogenic egg chambers and unfertilized eggs (Coelho et al., 2021; Pascual et al., 2022) the number of Calpains that are expressed is small. Noteworthy is Rp-CalpainA/B2, expressed at significant levels in vitellogenic oocytes. In early embryos (0–48 h), low expression levels are detected for Rp-CalpA/B1, Rp-CalpA/B2 and the non-classical Rp-CalpD and Rp-Calp7 (Table S2).

We also examined expression in fat body, testis, antennae, nervous system and gut transcriptomes. Surprisingly, only Rp-Calp7 is expressed in the gut, based on public RNAseq libraries available, the organ in which Trypanosoma parasites differentiate. Finally, non-classical Calpains are expressed at high levels in the antennae, and all R. prolixus Calpains are expressed to some extent in the nervous system. Therefore, all the R. prolixus calpain loci herein identified seem to be functional, with regulated gene expression in different developmental stages and tissues.

Next, we performed calpain knockdown (KD) using double-stranded RNA injected into the female abdomen (parental RNAi). Considering previously reported roles for Calpain loci in Drosophila oogenesis and embryogenesis (Kokai et al., 2012; Fontenele et al., 2013; Vieira et al., 2017), we were interested in investigating whether these roles are conserved in R. prolixus. Based on the phylogenetic and gene expression analysis we selected 7 loci for parental RNAi: Rp-CalpA/B1, Rp-CalpA/B2, Rp-CalpA/B3, Rp-CalpA/B4, Rp-CalpC, Rp-CalpD and Rp-Calp7. The number of eggs produced by each injected female was scored to define fecundity. We observed a significant decrease in oviposition after Rp-CalpA/B1, Rp-CalpC and Rp-CalpD knockdowns, compared to control (Fig. 4). Accordingly, few choriogenic oocytes are observed in Rp-CalpD KD, indicating halted oogenesis during the vitellogenic stage (Fig. 4B). Similar ovarian phenotypes where not observed for Rp-CalpA/B1 and Rp-CalpC KDs due either to more subtle effects on oogenesis or to effects during earlier stages. Curiously, Rp-CalpA/B4 KD laid to an increase in egg lay that was not explored further (Fig. 4D,E). Despite the absence of a detectable effect on oogenesis and egg laying, close examination of the microfilament network in the thropharium, which provide the cells and biochemical components for growing egg chambers, is disrupted in Rp-CalpA/B2 KD (Fig. S4).Fig. 4 R. prolixus Calpains play a role in oogenesis and embryogenesis. Functional analysis of Rp-Calpains was performed by parental RNA interference. Shown in (A-D) are ovaries dissected from (A) control GFP; (B) Rp-CalpD; (C) Rp-CalpA/B2 or Rp-CalpA/B4 KDs, 8 days after dsRNA injections. (B) Represents the entire ovary of an injected female, with a reduced number of eggs when compared to the eggs from the entire ovary in the control (A). Rp-CalpA/B4 KD ovaries seem to display delayed egg maturation, which may explain the apparent increase in egg number, as defined by the narrow collection window. tr=tropharium, pv=previtellogenic egg chambers, v=vitellogenic egg chambers, ch=choriogenic egg chambers. (E) Fecundity of injected females, defined as eggs laid per day per female. (F) Fertility of injected females, defined as the percentage of eggs that hatched as first instar nymphs. *p ≤ 0.05,**p ≤ 0.01.

Fig 4

Albeit parental knockdown is frequently used to explore the effects of maternal transcripts on the embryo, it also alters gene expression during embryogenesis itself since double stranded RNAs are transferred to developing embryos (Bucher et al., 2002; Zhu et al., 2020). We observed a significant decrease in embryo viability after Rp-CalpA/B2 and Rp-CalpD KDs compared to control (Fig. 4F), although it is yet unclear whether this results from a maternal or a zygotic effect of the knockdown. Double-stranded RNA injections for other Calpain loci had no effect on embryonic development. The oogenesis and embryonic phenotypes observed for Rp-CalpA/B4 and Rp-CalpD KDs are consistent with a decrease in mRNA levels during oogenesis (Fig. S5).

3.3 Rp-CalpA/B2 knockdown disrupts embryonic patterning

Since we found a significant decrease in embryo viability after Rp-CalpA/B2 and Rp-CalpD knockdowns, we asked whether these resulted from developmental abnormalities. Initially, we looked at stage 5 embryo morphology, when wild type embryos have completed gastrulation and initiated segmentation (Berni et al., 2014). We did not detect gastrulation disruption or arrested development at this stage in Rp-CalpD KD embryos (Fig. S6C), indicating that loss of viability does not result from defective embryonic patterning or early morphogenetic events. In contrast, we noted a reduced number of gastrulating embryos after Rp-CalpA/B2 knockdown, with approximately 90 % penetrance (Fig. 4F).

To investigate whether the embryonic loss of viability Rp-CalpA/B2 KD phenotype is due to early effects on embryonic patterning, such as those reported for D. melanogaster CalpA on the embryonic DV axis, we performed in situ hybridization for dorsal-ventral patterning genes. In Drosophila, loss of CalpA function results in a narrow ventral mesoderm domain, due to altered Toll pathway responses (Fontenele et al.,. 2013). In R. prolixus, knockdown for Toll pathway genes also leads to loss of the mesoderm as well as loss of expression of the mesodermal marker Rp-twist (Berni, Mota et al., 2023). Therefore, to investigate a putative function for Rp-CalpA/B2 in R. prolixus DV patterning and, more specifically, mesoderm formation, we looked at the Rp-twist expression pattern. Rp-twist marks a triangular shaped ventral expression domain during blastoderm stages, that moves posteriorly with the embryonic rudiment (Berni, Mota et al., 2023) (Fig. 5). We observed a dorsal expansion of the Rp-twist gene expression territory in Rp-CalpA/B2 KD embryos compared to control (Fig. 5C,D). Although the placement of the twist domain along the anterior-posterior axis follows the control pattern, it extends to lateral and dorsal regions of the embryo with high frequency. During germband extension stages, Rp-twist is expressed in the central region of the germband, and in regions that will give rise to the appendices (Berni, Mota et al., 2023). In contrast, germband extension was entirely blocked in Rp-CalpA/B2 KD embryos, with Rp-twist expression observed in a localized group of cells at the most posterior region of the embryo (Fig. 5E,F, Fig. S6B). This pattern indicates that these cells failed to undergo gastrulation.Fig. 5 Rp-CalpA/B2 knockdown alters dorsal-ventral patterning.Rp-soxN (A,B) and Rp-twi (C-F) expression revealed by whole mount in situ hybridization of stage 2 (A.B), 3 (C,D) or 5 (E,F) embryos from mothers injected with control (A,C,E) or Rp-CalpA/B2 dsRNA (B,D,F). Both Rp-soxN and Rp-twi are expanded dorsally in the Rp-CalpA/B2 KD. Figures in (A’,B’) are DAPI stains of (A,B). (G,H) Distribution of dorsal expansion phenotypes for Rp-twi (G) and Rp-soxN (H) in Rp-CalpA/B2 KD. n = 18 and n = 17 for Rp-CalpA/B2 KD embryos stained for stage 2 Rp-twi and stage 3 Rp-soxN, respectively.

Fig 5

The lateral neuroectodermal territory is also displaced in Rp-CalpA/B2 KD. The neural marker Rp-soxNeural (Rp-soxN) was previously identified in the R. prolixus genome, and in situ hybridization data shows it is a bona fide lateral marker that shows two stripes of expression during late blastoderm stages (Berni, Mota et al., 2023). In CalpA/B2 KD we observed an expansion of the Rp-soxN expression territory with high frequency (Fig. 5A,B). These results indicate that halted gastrulation observed in the Rp-CalpA/B2 knockdown is due to extensive failure of DV patterning during blastoderm stages.

4 Discussion

4.1 The R. prolixus Calpain system

Calpains are present in all living organisms, from bacteria to higher eukaryotes (Spinozzi et al., 2021). Ancestral Calpains were likely composed of MIT-CysPc-CBSW and EF-Hand domains, some of which were lost and shuffled with additional domains to generate ancestral CysPc-CBSW-EF-Hand classical Calpains (such as Calp A/B variants and CalpC) and CysPc-CBSW plus variable domain containing non-classical Calpain proteases (such as CalpD and Calp7). Accordingly, members of these subfamilies are arranged as a monophyletic clade in the phylogeny of eukaryotic supergroups (lower eukaryotes, invertebrates and vertebrates), subsequently diverging as the two classical and non-classical Calpain branches (Maki et al., 2012; Zhao et al., 2012). The analysis of R. prolixus Calpain sequences herein undertaken conforms to this distribution, as Calpains A/B and CalpC (classical Calpains), versus CalpD and Calp7 (non-classical Calpains) are distributed as different subgroups containing proteins with the expected domains. Interestingly, our study supports a great expansion of Rp-CalpA/B genes, with seven representatives in the kissing bug, as compared to non-hemipteran species investigated in this report and by others (Sorimachi et al., 2011; Henriques et al., 2017). In the future, it will be interesting to investigate additional related species to define whether this Calpain A/B expansion is restricted to R. prolixus or represents a pattern characteristic of the hemiptera order.

Our phylogenetic analysis also reinforces and extends previous findings suggesting the loss of Calp7 exclusively in flies (Sorimachi et al., 2011). We identified several insect Calpains containing the modular MIT-CysPc-CBSW structure characteristic of Calp7 (Maki et al., 2012), with a conserved catalytic triad in the CysPc and residues required for protein-protein interactions and membrane-anchoring functions in the CBSW domain. Accordingly, Rp-Calp7 (RPRC014368) contains an MIT domain at the N-terminus, as expected from a typical Calp7 domain architecture. On the other hand, despite the presence of an MIT domain at the C-terminus, C. lectularius CalpA/B (CLEC006431) and Z. nevadensis CalpA/B (XP021940249.1) were sub-clustered in the CalpA/B tree. This may be explained by the wide distribution of the MIT domains among eukaryotes, that indicates that it may have been present since very early stages of Calpain evolution, before the divergence of classical and non-classical Calpains (Maki et al., 2012; Zhao et al., 2012). The MIT domain is likely required for conserved subcellular functions such as that exerted in human Calpain 7, where it is required to interact with a subset of endosomal proteins for transport through the endosomal sorting complex (ESCRT) (Osako et al., 2010).

4.2 Catalytically inactive insect Calpains

The most surprising observation revealed by our phylogenetic analysis was the recurring identification of Calpains devoid of catalytic residues in several sub-branches of the insect CalpA/B tree. The absence of one or more residues of the catalytic triad has been used as a hallmark for the lack of catalytic activity, based on functional as well as structural data. Calpains with mutated catalytic residues have been produced by classical mutagenesis for murine Calpain 1 and Calpain 2. In these assays, the authors disrupted the active site Cys>Ser to generate a non-autocleavable sample of Calpains for crystallographic analysis (Pal et al., 2003; Hanna et al., 2008). Similar assays were performed for the unconventional Calpain 3, which requires Cys>Ala modification for complete loss of proteolytic activity (Ye et al., 2018). Importantly, CysPc harbors the catalytic triad residues, formed by Cys, His, and Asn, with a Trp residue stabilizing the His in the active site and being critical for Calpain activity (Pal et al., 2003). Therefore, it is reasonable to use the loss of these residues to imply loss of catalytic activity. Non-proteolytic Calpain homologues were previously described for different clades, including vertebrate CAPN6, Drosophila CalpC, C.elegans and protozoan calpain homologues (Friedrich et al., 2004; Sorimachi et al., 2010).

Calpains lacking catalytic residues were herein identified in the classical CalpC and CalpA/B branches. Since all species had one sole CalpC homologue, these probably arose as catalytically inactive Calpains at the basis of the CalpA/B and CalpC divergence. Contrarily, calpains lacking catalytic residues appear more than once in the CalpA/B tree, suggesting independent events. This has been confirmed by two different methods of tree building, using the full protein sequence or restricted to the CysPc domain. The detection of a monophyletic group comprised solely of CalpA/B sequences with substitutions at catalytic residues of the CysPc active site suggests that this branch arose as an ancestral CalpA/B duplication event, having lost catalytic residues in an ancestor of hemi- and holometabola. On the other hand, catalytically inactive R. prolixus CalpA/B proteins were probably generated by a second duplication event with loss of catalytic residues, since they are part of an independent branch (Rp cluster).

In principle, the loss of catalytic residues could lead to Calpain proteins that have lost their functional significance as modulatory proteases. However, other functions have been reported for Calpains. For instance, in protozoans that display a great Calpain expansion, dead- Calpains contribute to essential stages of the parasite's life cycle (Ennes-Vidal et al., 2021) and are associated with drug resistance (Vergnes et al., 2007). Accordingly, whereas the function of catalytically inactive D. melanogaster Calpain C is far from fully understood, it is highly expressed in salivary glands (Spadoni et al., 2003), in the adult midgut epithelium (FCA -snRNA-seq public data), in the ovary and larval imaginal discs, and expression increases after Sindbis virus infection (data available in FlyBase). Physically, D. melanogaster Calpain C interacts with Calpain A, B and TBPH (human TDP-43) (BioGRID, Oughtred et al., 2021), suggesting that CalpC is required either to control the enzymatic activity of catalytic active Calpain members, or functions as a scaffold protein. Herein we have shown that Rp-CalpA/B4 and Rp-CalpC knockdown decreases egg lay, which may suggest a role in oogenesis for their encoded catalytically inactive proteins. We also presented evidence that strongly indicates a role for one of the newly identified CalpA/B dead-Calpains (Rp-CalpA/B2) in R. prolixus early embryogenesis. Collectively, these results indicate that catalytically inactive members of the Calpain family have significant biological roles that need to be further dissected.

4.3 The R. prolixus “dead” Calpaina/B2 regulates embryonic patterning

To investigate early developmental functions for R. prolixus Calpains we performed parental knockdown assays for seven loci that displayed expression during oogenesis and/or early embryogenesis. We observed oogenesis (Rp-CalpA/B1, Rp-CalpA/B4, Rp-CalpC, Rp-CalpD) and/or embryogenesis (Rp-CalpA/B2 and Rp-CalpD) defects resulting from the KDs. However, functional redundancy and ineffective knockdown may have hampered the identification of additional effects. Loss of embryonic viability was clear and reproducible for Rp-CalpA/B2 and Rp-CalpD KDs, but only Rp-CalpA/B2 KD resulted in morphogenetic defects. These effects are most likely due to a decrease in Rp-CalpA/B2 messages either in the tropharium or in the early embryo (Table S2), since expression levels do not drop in Rp-CalpA/B2 KD ovaries enriched in vitellogenic and choriogenic egg chambers (Fig. S5). Alternatively, they may result from indirect effects of the KD in other tissues.

in situ gene expression data indicates that loss of embryonic viability in Rp-CalpA/B2 KD results from early defects in axial patterning and consequent Inability to extend the germband. Previously, we explored the role of Toll pathway (Berni et al., 2014), and BMP pathway (Berni, Mota, et al. 2023) elements in R. prolixus embryonic development. Germband extension was blocked in both Rp-dl KD (Toll effector) and Rp-dpp or Rp-gbb KD (encoding BMP ligands), resulting from defects in dorsal-ventral patterning during the blastoderm stages (Berni, Mota, et al., 2023). Here we have shown that ventral and lateral gene expression is expanded dorsally in blastoderm stage Rp-CalpA/B2 KD embryos, closely resembling the pattern we observed with BMP pathway loss-of-function assays. Interestingly, CalpA loss-of-function alters the extent of ventral domains in the Drosophila embryo as well (Fontenele et al., 2009 and 2013). These results suggest a conserved role of Calpain A/B like genes to establish axial patterning in a dipteran (D. melanogaster) and a hemipteran (R. prolixus). This is remarkable, considering that DV patterning in Drosophila relies mostly on the Toll pathway to establish the DV axis, while in R. prolixus it is the BMP pathway that performs a major role in this context (Reeves and Stathopoulos, 2009; Roth et al., 2023; Berni, Mota et al., 2023). Consequently, one is left to answer whether the mechanisms deployed for Calpain function in early Drosophila and Rhodnius DV patterning are conserved.

To explore the possibility of conserved mechanisms of Calpain function between Drosophila and Rhodnius it is important to first consider which might be the Calpain target(s) in the early embryo. Both Dm-CalpA and Rp-CalpA/B2 are expressed during the early stages of embryonic patterning and KD for either locus results in DV patterning defects. However, Dm-CalpA KD leads to loss of ventral Dm-twist gene expression while Rp-CalpA/B2 KD leads to the expansion of ventral gene expression such as seen for Rp-twist. One possible explanation for this discrepancy is the absence of enzymatic activity displayed by Rp-CalpA/B2, which could result in binding but not cleavage of a conserved Calpain target (such as the Cactus/IκB inhibitor, CalpA target in D. melanogaster; Fontenele et al., 2013). An alternative hypothesis would state that CalpA and Rp-CalpA/B2 target completely different and undescribed proteins that impact Dm-twist or Rp-twist expression in the ventral prospective mesoderm. While the first hypothesis is testable by co-immunoprecipitation, once specific anti-Cactus antibodies are developed for the kissing bug, the second would require characterization of the repertoire of Calpain targets in both species. Unfortunately, it is not possible to test the hypothesis of Rp-Cact as an embryonic Rp-CalpA/B2 target in double KD experiments since parental KD for Rp-cact results in loss of fecundity (Berni et al., 2014).

With respect to the hierarchical relationships between CalpA or Rp-CalpA/B2 and the Toll and BMP signaling network that establishes DV patterning in D. melanogaster versus R. prolixus, several open questions remain (Fig. 6). The epistatic relationships between the Toll and Dpp pathways in early Drosophila embryogenesis have been extensively explored. The Toll pathway induces ventral expression of twist and lateral expression of the BMP inhibitor encoded by sog, but inhibits dpp expression in these regions (Rushlow and Shvartsman, 2012; Stevens and Stein, 2014; Schloop et al., 2020). In parallel to the Toll pathway, CalpA binds the Cactus/IκB inhibitor and is required to establish high levels of nuclear Dl that are required to induce the most ventral fates, as characterized by twist expression (Fig. 6A; Fontenele et al., 2013). Interestingly, maternal dpp inhibits CalpA expression and activity, while the Toll pathway has no effect (Fontenele et al., 2009; Araujo and Bier, 2000). In R. prolixus, we have shown that the Toll pathway NFκB superfamily effector encoded by Rp-dl increases Rp-sog expression (Berni and Mota et al., 2023), which in turn establishes a BMP gradient that restricts Rp-twist expression to the ventral domain of the embryo (Fig. 6B). Assuming that Rp-CalpA/B2 interacts with the NFκB inhibitor Rp-Cactus, based on the fact that Cactus/IκB proteins are evolutionarily conserved Calpain targets, Rp-CalpA/B2 could function to bind Rp-Cactus, leaving Rp-Dl free for nuclear translocation to enhance Rp-sog expression. In the absence of Rp-CalpA/B2 (in the knockdown), free Rp-Cactus would inhibit Rp-Dorsal, resulting in less Rp-sog expression, and loss of polarized Rp-Dpp that is required to restrict Rp-twist expression ventrally (Fig. 6B). However, Rp-CalpA/B2 KD expands Rp-twi completely while in Rp-sog KD the Rp-twi domain is expanded only to half the embryo (Berni, Mota et al., 2023), indicating that Rp-CalpA/B2 modifies additional targets or that it acts through a completely different mechanism in the kissing bug. Currently, it is unclear whether Rp-Cact is a Rp-CalpA/B2 substrate and whether Rp-Toll or Rp-Dpp control Rp-CalpA/B2 expression in the context of kissing bug embryogenesis. Considering the changing roles of these upstream elements of the insect DV patterning network, it will be interesting to investigate whether the Rp-Toll or Rp-BMP pathways exert any effect on Rp-CalpA/B2 function.Fig. 6 Schematic model for the epistatic relations between Calpains and the Toll and BMP pathways in Drosophila and Rhodnius DV patterning. (A) In D. melanogaster Toll plays a major role in DV patterning, establishing the entire DV axis, while BMPs role is restricted to patterning the dorsal part of the embryo. By inducing the nuclear translocation of the NFκB/c-Rel protein Dorsal (nDl) it induces the ventral and lateral expression of genes such as twi and sog, and inhibits dpp expression, restricting BMPs dorsally. Calpain A binds and cleaves the IκB Cactus inhibitor (not shown), favoring Dorsal nuclear translocation in ventral (Toll high) regions of the embryo. Toll signals have no effect on Calpain A expression or protein activity but Calpain A is inhibited by the BMP encoded by dpp. (B) In R. prolixus the BMP pathway plays a major role in DV patterning, while Toll only polarizes (dotted arrow) the BMP modulator encoded by sog. Ventrally produced Sog protein establishes graded BMP by shuttling BMP protein dorsally, thus defining the position of ventral and lateral domains of the embryo. Without BMPs, ventral (Rp-twi) gene expression expands dorsally. Rp-Toll has no effect on Rp-dpp expression. Rp-CalpA/B2 KD expands the ventral Rp-twi domain dorsally, suggesting it modifies the Toll (interrogation mark) pathway as in Drosophila and/or the BMP pathway. It has not been established whether Rp-CalpA/B2 regulates R. prolixus IκB/Cactus and whether the Toll or BMP pathways regulate CalpA/B2 expression.

Fig 6

Notably, Drosophila CalpA and R. prolixus CalpA/B2 proteins display substantial differences in domain-structure architecture, such as the absence of active catalytic residues and a PEF domain in Rp-CalpA/B2. Functional assays in the Drosophila embryo suggest that CalpA, in addition to its catalytic function, also controls subcellular distribution and availability of its targets (Fontenele et al., 2013; Julio et al., unpublished results), suggesting that active and inactive Calpains could share some common mechanisms. Even though, to fully understand the mechanism of Rp-CalpA/B function it will be necessary to examine the protein interactome for R. prolixus CalpA/B2 and D. melanogaster CalpA during embryogenesis. In conclusion, despite the differences in the DV patterning signal transduction network between Drosophila and Rhodnius, our analysis of the role of Calpains in R. prolixus suggests a conserved role for CalpA/B encoding loci in insect early embryogenesis, which is not exclusively associated with its enzymatic activity.

Author contributions

H.A and A.J. conceived the study and designed the experiments. H.A and P.M.B supervised the research. A.J performed Calpain identification, phylogenetic and structural analysis. AJ and TG performed functional assays and quantification. MB developed tools for functional analysis. A.J and H.A wrote the paper with input from all authors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Image, application 2

Data availability

All data is available in the main text or in supplementary files. A datasheet with raw processed data is provided.

Acknowledgements

We would like to thank members of the Araujo lab for helpful discussions. We are grateful to the animal facility at the Institute of Medical Biochemistry for technical assistance with Rhodnius husbandry. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, E26/010.001/795/2019 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 88881.117632/2016–01 ) to HA, and FAPERJ grant (FAPERJ, E-26/010.001242/2016 ) to PMB. HA and PMB are CNE FAPERJ researcher and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) fellows. AJ and MB were supported by post-graduate fellowships from CAPES. AJ was also supported by post-graduate FAPERJ-Nota10 fellowship. TG was supported by a pos-doctoral fellowship from CNPq.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cris.2024.100094.
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