Comparison of the Malpighian tubules and fat body transcriptional profiles of Zophobas morio larvae (Coleoptera: Tenebrionidae)

Comparative Biochemistry and Physiology - Part D 29 (2019) 95–105

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Comparison of the Malpighian tubules and fat body transcriptional profiles of Zophobas morio larvae (Coleoptera: Tenebrionidae) Jaqueline R. Silvaa,b, Danilo T. Amarala, Vadim R. Viviania,b, a b

T

⁎

Graduate School of Biotechnology and Environmental Monitoring (UFSCar), Sorocaba, SP, Brazil Graduate School of Evolutive Genetics and Molecular Biology, (UFSCar), São Carlos, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: AMP-CoA ligases Detoxification Excretion Coumarate-like enzyme Transcriptome

The Malpighian tubules in insects play an essential role in osmoregulation, through the transport of ions during excretion, whereas the fat body is usually associated with the intermediary metabolism. The tubules also are involved in excretion of organic solutes and xenobiotics. However, with the exception of a preliminary transcriptional survey of the Zophobas morio (Tenebrionidae) larval tubules, there are no detailed transcriptional analysis of this organ in Coleoptera. A luciferase-like enzyme that displays weak luminescence activity in the presence of firefly D-luciferin and ATP was cloned from the tubules of Z. morio larvae. In order to better understand the molecular physiology of Malpighian tubules and fat body in Coleoptera larvae, and to investigate the occurrence and functions of AMP-CoA ligases in these tissues, we performed a comparative transcriptional analysis of these tissues using Z. morio giant-mealworms. As expected, the tubules displayed organic and inorganic transporters, xenobiotic metabolism enzymes, V-ATPases, channels, and pumps. The fat body showed proteins that are synthesized in this tissue and secreted to the hemolymph, as well as enzymes involved in lipid and carbohydrate metabolism. These tissues are also involved in common pathways, such as nitrogen metabolism to degradation/excretion, eye pigments biosynthesis, immunity, and detoxification. The presence of coumarate-CoA ligase-like enzymes in these tissues suggest their involvement in the degradation of coumaric acid derivatives obtained from the diet, or alternatively, in the biosynthesis of compounds structurally related to coumaric acids such as eye pigments. Our results confirm to the physiological versatility of tubules and fat body in larval Coleoptera.

1. Introduction The osmoregulation and excretion in insects, which is essential for the survival of such small animals in a wide range of habitats and diets, are performed by the Malpighian tubules and rectum. The Malpighian tubules produce the primary urine during the transepithelial transport of ions and other compounds from the hemolymph to the lumen. After the primary urine formation in the tubules, water and other useful compounds are conserved by the rectum through reabsorption, and the remnant fluid is excreted (Wigglesworth, 1972; Maddrell and Gardiner, 1976). A transcriptional analysis of Drosophila tubules showed other roles in addition to ions and water transport (Wang et al., 2004). The tubules perform the excretion of several toxic compounds, such as ouabain (Torrie et al., 2004), salicylate (Ruiz-Sanches et al., 2007), cardiac

glycosides (Rafaeli-Bernstein and Mordue, 1978), nicotine, vinblastine (Gaertner et al., 1998), morphine and atropine (Maddrell and Gardiner, 1976), etc. Besides the general function of excretion of organic solutes, waste compounds, and xenobiotics, the Malpighian tubules may also be involved in other unusual functions in different insects, including the bioluminescence of Arachnocampa spp. (Diptera), in which the photogenic tissues consist of modified terminal ends of the tubules (Gatenby, 1960; Viviani et al., 2002), and the tubules of some Neuroptera and Diptera larvae which produce silk (Wigglesworth, 1972). Previously, a luciferase-like enzyme was cloned from the tubules of Zophobas morio larvae. This enzyme is a CoA-ligase that displays weak luminescence activity with ATP and firefly D-luciferin, a xenobiotic for this non-bioluminescent insect (Viviani et al., 2009). AMP-CoA ligases, or adenylate-forming enzymes, are a large family of enzymes with

Abbreviations: ABC, ATP binding cassette; CoA, coenzyme A; EDTA, ethylenediamine tetraacetic acid; FPKM, Fragments Per Kilobase Million; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NCBI, National Center for Biotechnology Information; WEGO, Web Gene Ontology Annotation Plot ⁎ Corresponding author at: Graduate School of Biotechnology and Environmental Monitoring (UFSCar), Sorocaba, SP, Brazil. E-mail address: [email protected] (V.R. Viviani). https://doi.org/10.1016/j.cbd.2018.11.007 Received 11 October 2018; Accepted 7 November 2018 Available online 09 November 2018 1744-117X/ © 2018 Published by Elsevier Inc.

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2. Materials and methods

number). The transcripts abundance in each tissue was compared using FPKM values (Fragments Per Kilobase Million), and calculated using the align_and_estimate_abundance.pl script which is included at Trinity. Selected amino acids sequences were aligned using the Clustal W algorithm tool (Higgins et al., 1994) in MEGA 6.0 software (Tamura et al., 2013). Phylogenetic analysis was carried out using the software MrBayes 3.2 (Ronquist et al., 2012), and the best evolutionary model (LG + G + I + F) was predicted using ProtTest (Abascal et al., 2005). MrBayes analyses were run twice for 10,000,000 generations, sampling trees each 1000 generations. We discarded the first 25% of trees and concatenated the remaining to create the consensus tree, using posterior probabilities as branches supported values. The tree was visualized using the software FigTree v.1.3.1 (Rambaut, 2007).

2.1. Zophobas morio rearing and maintenance

2.5. PCR analysis of AMP-CoA ligases sequences

Larvae of Zophobas morio were obtained from the vivarium of UNESP - Rio Claro (São Paulo State University “Júlio de Mesquita Filho”) and reared on wheat bran and cassava flour at room temperature in darkness.

The total RNA from Malpighian tubules and fat body were used to synthesize the corresponding cDNA using High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, United Kingdom). The obtained cDNAs were used as templates for amplifying sequences by PCR with CoA-ligase specific primers (Table A.1). These primers were designed from transcriptome sequences that are similar to AMP-CoA ligases enzymes (~40% of identity). PCR reactions were performed using PCR Master Mix kit (Promega, USA) and PCR conditions were: (1) initial denaturation cycle of 5 min at 95 °C, (2) 30 cycles of 1 min at 95 °C (denaturation), 1 min at 55 °C (annealing) and 1 min at 72 °C (extension), and (3) a final step of 10 min at 72 °C. Amplification results were analyzed by 1% (w/v) agarose gel electrophoresis in TAE 1× (20 mM Tris-acetate and 0,5 mM EDTA) buffer and visualized by fluorescence using UV transilluminator.

distinct biological functions such as fatty acid activation, polyketide biosynthesis, pigment biosynthesis and xenobiotic detoxification, which activate carboxylic acid through ATP-dependent adenylation and usually followed by thioesterification to CoA (Viviani, 2002; Schmeiz and Naismith, 2009). With exception of beetle luciferases, studies about enzymes of this family in insects are still missing. Considering that the physiology of the Malpighian tubules and fat body in beetles and their larval stages are far from being fully understood, here we performed the first comparison of the transcriptional profiles for the Malpighian tubules and the fat body for a model of Coleoptera larva, the giant mealworm Zophobas morio (Tenebrionidae).

2.2. Total RNA extraction, cDNA library preparation and Illumina sequencing Malpighian tubules and fat body of Zophobas morio larvae were isolated with fine forceps in insect physiological buffer (12 mM NaCl, 1.3 mM Na2PO4, 1.2 mM KH2PO4). After isolation, the tubules and fat body were transferred to ice-cold TRIzol reagent (Life Technologies, USA) for total RNA extraction following the manufacturer instructions. The quality/quantity of total RNA extractions was spectrophotometrically determined using NanoView spectrophotometer (GE Health, USA), and it was also measured by Agilent 2100 Bioanalyzer (Agilent Tech., USA). The mRNA isolation and the cDNA library construction from both tissues were performed at LaCTAD facility (UNICAMP, BR), using TruSeq RNA Sample Preparation Kit (Illumina, Inc., USA). The tagged cDNA libraries were pooled in equal ratios and used for 2 × 150 bp paired-end sequencing on a single lane of the Illumina HiSeq2500, according to the manufacturer instructions (Illumina, Inc., USA). The paired-end reads are available at the SRA (Sequence Read Archive) on NCBI database (project number: PRJNA400859).

3. Results and discussion 3.1. De novo transcriptome assembly of Malpighian tubules and fat body The Malpighian tubules and fat body cDNA libraries were sequenced, resulting in ~120 and ~45 million raw reads, respectively. After data processing and de novo assembly, we obtained 66,698 contigs from tubules and 91,799 from fat body. From them, we obtained 22,242 non-redundant transcripts for tubules and 18,589 for fat body after overlapping sequences that displayed > 95% of similarity.

2.3. De novo transcriptome assembly 3.2. Functional annotation The sequencing quality was checked using FastQC 0.11.5 tool (Andrews, 2010). Adaptors and low-quality reads (Phred Q ≤ 30) were removed using FASTX-TOOLKIT 0.013 (Pearson et al., 1997). The cleaned reads were de novo assembled using Trinity 2.0.2 software, which combines three independent modules: Inchworm, Chrysalis, and Butterfly (Grabherr et al., 2011), on the DIAG Academic Grid at University of Maryland. The contigs were translated to amino acid by TransDecoder tool presents at Trinity, with overlapping sequences that displayed > 95% of similarity (Unigenes or transcripts).

Blast hits and annotation of the assembled transcripts from both tissues are summarized at Table 1. Several transcripts did not display any similarity with the database (3559 to tubules and 2268 to fat body), indicating that many genes/proteins are still unknown, and may perform important functions in these tissues. As expected, the annotation hits in terms of species distribution were similar to gene products of the closely related species Tribolium castaneum (Coleoptera: Tenebrionidae). GO terms, KEGG pathway, and InterPro annotations for tubules and fat body transcripts were performed by Blast2GO software. Tables A.2 and A.3 exhibit detailed data of functional annotation of Malpighian tubules and fat body, respectively. These supplementary files show

2.4. Transcript annotation, computational analysis of sequences and abundance calculation All assembled unigenes were subjected to similarity search against NCBI non-redundant (nr) and UNIPROT/SWISS-PROT databases using BLASTp algorithm on Blast2GO software (Conesa et al., 2005), with a cut-off E-value of ≤10−5. Following the Blast2GO mappings steps, the Gene Ontology (GO) terms search was performed with E-value < 10−6, annotation cut-off > 55, being graphically expressed using Web Gene Ontology Annotation Plot (WEGO) tool (Ye et al., 2006). The pathways annotations were also determined using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Enzyme Commission Numbers (EC

Table 1 Blast hits and annotated sequences of each analyzed tissue. Database NCBI SwissProt

96

Blast hits Annotation Blast hits Annotation

Malpighian tubules

Fat body

18595 contigs 8782 contigs 14,311 contigs 10,908 contigs

16320 contigs 7996 contigs 13,295 contigs 9178 contigs

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several information about each transcript, such as description, lenght, e-value, GO categories, enzymes codes, and InterPro IDs (Phobius, Pfam). GO categories are classified in three categories: molecular function, biological process and cellular component. For tubules, 8782 transcripts were assigned GO terms, and 7996 transcripts for fat body (Fig. A.1). KEGG pathway analysis showed that the transcripts from tubules are involved in 112 pathways, and those from fat body in 114. The pathways with most representation in both tissues were from purine metabolism, thiamine metabolism, and antibiotics biosynthesis (Tables A.4 and A.5). The analyzed tissues are capable of metabolizing purinic compounds to excretion (Nation, 2015), therefore, it is expected to find transcripts involved in purine metabolism. In this pathway, uric acid from degradation of nucleic acids and proteins is produced mainly in the fat body and is precipitated in the Malpighian tubules for storage or excretion (Nation, 2015). In relation to thiamine metabolism, the tissues showed three enzymes families (desulfurases, phosphatases, diphosphokinases). The phosphatases and diphosphokinases catalyze the formation of the phosphate intermediaries of thiamine. Thiamine pyrophosphate is a co-factor in many reactions, such as in the conversion of succinyl-CoA in the tricarboxylic acid cycle for energy generation in cells. Insects require thiamine as a vitamin (B6) from their diets (Candy, 1985), and Z. morio larvae have a thiamine rich diet from wheat bran.

Table 3 Most abundant transcripts in fat body of Z. morio larvae obtained in this study.

The fifteen most highly abundant transcripts in tubules and fat body are shown in Tables 2 and 3, respectively. In comparison, glyceraldehyde-3-phosphate, a housekeeping gene, exhibited the following abundance values: 410,26 in tubules and 231,47 in fat body, in FPKM. Some abundant transcripts from Malpighian tubules are uncharacterized or unknown yet, and others have their functions predicted, such as glutathione S-transferase sigma, which is a common class found in insects and it has some housekeeping-related roles being also involved in insecticide metabolism (Liu et al., 2014). Although the functions of takeout/juvenile hormone binding proteins, synaptic vesicle glycoprotein 2b, and lazarillo protein remain unknown in tubules, they may play important roles, since they are the most abundant transcripts in this tissue. In Aedes aegypti tubules of larvae (aquatic habitat) and adults (blood feeders), the most abundant transcripts were similar to cytochrome c oxidase subunit I and some unknown proteins (Li et al., 2017). Similar to A. aegypti tubules of adults (Li et al., 2017),

Description

comp13348_c1_seq1 comp14647_c0_seq1 comp15982_c3_seq1 comp15764_c0_seq1 comp7348_c0_seq1 comp17006_c0_seq1 comp19246_c3_seq1 comp15826_c0_seq1 comp13348_c1_seq2 comp19221_c1_seq1 comp13436_c0_seq1 comp20473_c0_seq5 comp11790_c0_seq1 comp17324_c0_seq2 comp11847_c0_seq1 comp17324_c0_seq3 comp17324_c0_seq1 comp17114_c0_seq1 comp16400_c0_seq1

92,736.94 70,723.41 35,762.77 15,102.82 12,199.46 9064.49 7608.17 7546.96 6556.69 6264.35 5650.06 5317.06 5176.69 4437.16 4306.82 4053.87 3957.70 3303.65 3297.30

uncharacterized protein hypothetical protein uncharacterized protein synaptic vesicle glycoprotein 2b no similarity cytochrome c oxidase subunit 1 cytochrome c oxidase subunit 3 no similarity uncharacterized protein protein takeout uncharacterized protein takeout-like protein bifunctional nitrilase nitrile hydratase partial no similarity no similarity no similarity lazarillo protein-like glutathione s-transferase sigma transcriptional regulator atrx homolog

Description

comp6951_c0_seq1 comp11654_c0_seq1 comp15344_c0_seq1 comp16209_c0_seq2

52,837.30 44,150.17 36,223.89 31,186.27

comp16209_c0_seq1

22,250.28

comp14862_c3_seq1 comp14862_c0_seq1 comp12087_c0_seq1 comp14855_c2_seq1 comp10564_c1_seq1 comp14862_c1_seq1 comp14862_c2_seq1 comp13550_c0_seq1 comp16054_c1_seq1 comp12275_c0_seq2

21,795.48 19,660.74 18,537.55 17,112.12 15,411.23 15,180.35 12,062.51 11,995.51 11,967.84 10,810.08

apolipophorin-III precursor no similarity hexamerin 1B precursor 56 kDa early-staged encapsulation-inducing protein 56 kDa early-staged encapsulation-inducing protein hexamerin 2 precursor hexamerin 2 precursor hypothetical protein 28 kDa desiccation streess protein precursor 12 kDa hemolymph protein e precursor arylphorin precursor hexamerin 2 precursor 12 kDa hemolymph protein e precursor hexamerin 4 precursor no similarity

3.4. Transcripts of Malpighian tubules Most knowledge about mechanisms and physiology of tubules come from Drosophila melanogaster (Diptera) studies, allowing general inferences about the physiology of this tissue in insects due to the similarity of Malpighian tubules in this taxonomic group (Dow et al., 1994; Dow and Davies, 2001; Wang et al., 2004; Dow and Davies, 2006; Dow, 2009; Beyenbach et al., 2010). Transcriptional analysis are available for the tubules of Drosophila melanogaster (Wang et al., 2004), Anopheles gambiae - Diptera (Overend et al., 2015), Aedes albopictus - Diptera (Esquivel et al., 2016), and Tribolium castaneum – Coleoptera tubules, but in such study the libraries included both the tubules and hindguts (Park et al., 2008). With the exception of a preliminary transcriptional survey of the Z. morio larval tubules (Silva et al., 2015) no detailed transcriptomes or RNA-Seq studies were reported for this organ in Coleoptera. We identified some transcripts involved in ion transport machinery for urine production and normal operation of tubular cells, such as transporters, and proteins of cellular junctions (Table 4). Ions and organic solutes are transported to the lumen tubules by paracellular and transcellular pathways during the urine formation. Paracellular transport occurs between epithelial cells and it is controlled by septate junctions. On the other hand, transcellular pathway occurs via two cell membranes and involve membrane transporters (Chapman, 1998; Beyenbach et al., 2010). Vacuolar-proton-adenosine triphosphatase (VATPase) is the driving force for the ions movement during urine production, proton exchange and the generation of membrane voltages. The membrane voltages may contribute to transepithelial secretion and uptake of Na+ and K+ by principal cells, and Cl− secretion in stellate cells. Urine production also involves the transport of the other compounds to the lumen, such as uric acid, sugars, amino acids, and alkaloids (Chapman, 1998; Klowden, 2007; Beyenbach et al., 2010; Piermarini, 2016). Both Drosophila (Wang et al., 2004) and Z. morio

Table 2 Most abundant transcripts in Malpighian tubules of Z. morio larvae obtained in this study. FPKM

FPKM

Z. morio larvae also display ribosomal proteins with high abundance (data not shown). These data suggest that some transcripts possibly related to basic tubular function are abundant independently of insect order, environment, and feeding. The most abundant transcripts found in Z. morio fat body are similar to proteins or precursors that are synthesized in this tissue and secreted to the hemolymph, such as hexamerins, 28 kDa desiccation stress protein (dsp28) precursor, apolipophorin-III, and 12 kDa hemolymph (Table 3). The hemolymph dsp28 is involved in avoiding the desiccation damage (Graham et al., 1995), and the presence of this protein in Z. morio fat body may indicate its involvement in survival of the larvae in the dry environment where it is reared.

3.3. The most highly abundant transcripts in Malpighian tubules and fat body

Transcript ID

Transcript ID

97

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Table 4 Transcripts found in Malpighian tubules from Z. morio larvae involved in osmoregulation and homeostasis obtained in this study. The transcripts attributions were done based on functional annotation performed by Blast2GO software. Cellular and molecular mechanisms

Ion and water transport machinery to urine production

Proteins and Transcripts IDs V-ATPases

Subunits

comp18513_c0_seq1 comp17485_c0_seq1 comp12328_c0_seq1 comp14151_c0_seq1 comp13376_c0_seq1 comp19850_c0_seq2 comp10841_c0_seq1 comp15047_c0_seq1

A B C D E F G S1

Na+/K+ ATPase

Subunits

comp18373_c0_seq3 comp13293_c0_seq1 comp12464_c0_seq1

α β1 β2

448.01 382.13 255.16 228.79 787.12 49.48 3.90 246.51

43.15 47.90 2.95

Function

Translocate H+ from the cytoplasm to the lumen

Na+/K+ pump

NHE3 comp8478_c0_seq3

0.58

Coupled counter-transport of one H+ ion in exchange for one Na+ ion

Kir 2 comp20485_c0_seq3

19.96

Potassium channel

Chloride channel

CIC 2 comp20786_c0_seq1 comp20713_c0_seq4

Isoforms x1 x2

8.55 1.31

Aquaporins comp17011_c0_seq1 comp19301_c0_seq1 comp17186_c0_seq1

Isoforms AQPcic AQPAng.G NIP1-2

60.72 27.85 46.40

Carbonic anhydrase comp25187_c0_seq1

Transporters

FPKM

1.73

Sugar transporter comp22856_c0_seq1

2.22

Copper transporter comp17946_c0_seq1

140.72

Zinc transporter comp15113_c0_seq1

25.41

Folate transporter comp19687_c0_seq4

6.07

Glucose transporter comp19950_c0_seq5

14.18

Amino acid transporter comp19097_c0_seq1

27.95

Multivitamin transporter comp40208_c0_seq1

0.62

Treahalose transporter comp15819_c0_seq1

3.61

Water channel

H+ supply to V-ATPase

Organic and inorganic solutes transport

Transport

ABC transporters

Subfamilies

comp16524_c0_seq1 comp3025_c0_seq1 comp16801_c0_seq2

G14 G20 F4

11.02 1.05 20.84

(continued on next page)

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Table 4 (continued) Cellular junctions

Septate

Neuroglian comp20489_c0_seq2

9.32

Neurexin IV comp20164_c0_seq4

2.83

Contactin comp18375_c0_seq3

1.40

Lachesin comp18133_c0_seq1

7.27

Fasciclin

Adherens

GAP

Permeability barrier to solutes

Isoforms

comp16547_c0_seq1

II

6.94

comp15354_c0_seq1

III

5.23

Armadillo comp16749_c0_seq1

1.26

α-catenin comp20400_c0_seq1

7.65

Region of contact between adjacent epithelial cells

Innexin

Isoforms

comp15993_c0_seq1

1

5.15

comp14236_c0_seq1

2

40.71

comp17756_c0_seq2 comp17969_c0_seq1

3

5.25

7

22.42

Communication between neighboring cells

based on antimicrobial peptides synthesis, activation of enzymic cascades that regulate coagulation and melanization of hemolymph, and cellular responses that involve phagocytosis, nodulation, and encapsulation. Cellular responses are mediated by hemocytes while antimicrobial peptides production involve fat body cells that release them into hemolymph (Hoffmann, 2003; Attardo et al., 2006; Tsakas and Marmaras, 2010). However, other tissues, such as Malpighian tubules, are also involved in antimicrobial peptides production (Nappi et al., 2000). In Drosophila, the Toll and Imd pathways are involved in production of seven antimicrobial peptides (Hoffmann, 2003; McGettigan et al., 2005). The fat body of Z. morio larvae exhibited several transcripts related to immunity (Table A.6), such as lectins, and transcripts involved in innate immunity pathway and effectors. Comparatively, tubules transcriptome also indicated the presence of transcripts involved in immunity, such as toll receptor, spaetzle 3, tube (not found in fat body and related to Toll pathway), attacin, pelle, and cactus. Both tissues showed the Attacin peptide with low abundance, which act temporally in antimicrobial peptide synthesis.

larval (Table 4) tubules showed a wide range of organic and inorganic solutes transporters. The presence of several of them in tubules shows how important is the compounds transcellular transport in excretion in insects in general (Wang et al., 2004; Dow and Davies, 2006). 3.5. Transcripts of the fat body Transcriptional analysis of the fat body has already been carried out in many insects, such as Glossina morsitans morsitans - Diptera (Attardo et al., 2006), Camponotus floridanus - Hymenoptera (Gupta et al., 2015), Drosophila melanogaster (Jiang et al., 2005), Bombyx mori - Lepidoptera (Cheng et al., 2006), Aedes aegypti - Diptera (Price et al., 2013), Melipona scutellaris - Hymenoptera (Sousa et al., 2013), Bemisia tabaci Hemiptera (Wang et al., 2010), Tribolium castaneum - Coleoptera (Park et al., 2008), and luminescent beetles of Elateroidea superfamily in Coleoptera: Phrixothrix hirtus (Amaral et al., 2017a) and Aspisoma lineatum larvae (Amaral et al., 2017b). The results obtained here agree with these studies. We found and identified transcripts expected for the fat body, such as transcripts involved in immunity, storage and utilization of nutrients to energy generation, detoxification, nitrogen metabolism, and similar transcripts of proteins synthesized by the fat body cells and secreted to hemolymph, which were the most abundant transcripts in this tissue (Chapman, 1998; Klowden, 2007; Arrese and Soulages, 2010). Transcripts similar to proteins synthesized in the fat body, proteins related to cellular junctions in this tissue, and involved in metabolism of fatty acids and carbohydrates are shown in Table 5.

4.2. Detoxifying enzymes Malpighian tubules and fat body participate in detoxification of endogenous and exogenous compounds, such as insecticides. Besides this, tubules also excrete these compounds and it is well known that they display abundance of enzymes related to the detoxification metabolism, such as cytochromes P450, glutathione-S-transferases, alcohol dehydrogenase, ATP binding cassette (membrane transporters) (Wang et al., 2004; Dow, 2009; Chachine et al., 2012). Detoxification may also be carried out by hydrolases and carboxylesterases, which catalyze the hydrolysis of pyrethroids and organophosphates (Zhang et al., 2016). The fat body and tubules of Z. morio displayed transcripts identified as carboxylesterases.

4. Comparison between Malpighian tubules and fat body Although Malpighian tubules are involved mainly with excretion and osmoregulation (Wigglesworth, 1972; Chapman, 1998; Klowden, 2007) and the fat body is related to the intermediary metabolism in insects (Chapman, 1998; Klowden, 2007), both of them also play a role in immunity, eye pigment biosynthesis, detoxification, and nitrogen metabolism.

4.3. Cytochromes P450 Cytochromes P450-dependent monooxygenases are found in the endoplasmic reticulum and mitochondria of eukaryotes, and require NADPH. These enzymes participate in metabolism of endogenous and exogenous compounds by multiple oxidative reactions of their substrates. The number of cytochromes P450 isoforms found in the

4.1. Immunity Immune response in insects is originated mainly by hemocytes (hemolymph) and the fat body. They have an efficient immune system 99

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Table 5 Transcripts of the fat body from Z. morio larvae involved in cellular and molecular mechanisms obtained in this study. The transcripts attributions were done based on functional annotation performed by Blast2GO software. Cellular and molecular mechanisms

Proteins and Transcripts IDs

FPKM

Function

Cell-to-cell adhesion

Cellular junctions Desmosomes

Cadherin comp14657_c2_seq1 comp25403_c0_seq1

Isoforms 1 23

6.30 2.20

Hemidesmosomes

Integrin comp16699_c0_seq2 comp16509_c0_seq1

Isoforms α β

3.65 67.62

GAP

Innexin comp15546_c0_seq1 comp5754_c0_seq1 comp13737_c0_seq1 comp8763_c0_seq1

Isoforms 1 2 3 7

39.46 73.44 83.63 5.49

Proteins biosynthesis

Hexamerin comp15344_c0_seq1 Lipophorins comp15683_c0_seq1 Transferrin comp3656_c0_seq1 Juvenil hormone esterase comp11413_c0_seq1 Vitellogenin precursor comp25815_c0_seq1 12 kDa hemolymph comp13550_c0_seq1 28 kDa desiccation stress protein comp15313_c0_seq1

Lipid storage

Carbohydrate metabolism

Cell-to-extracellular matriz adhesion

Communication between neighboring cells

36223.89 767.02 108.28 11.52 1.53

These proteins are usually secreted to hemolymph and them play several functions

11995.51 17112.12

Diacylglycerol acyltransferase comp10683_c0_seq1 Lipid storage droplet 1 comp15408_c0_seq1 Phospholipase a comp19362_c0_seq1

Glycogen phosphorylase comp16330_c0_seq1 UDP-glucose pyrophosphorylase comp10716_c0_seq1 Phosphoglucomutase comp16542_c0_seq2 Trehalase

0.63 218.98 3.13

Triacylglycerol synthesis Protein found in lipidic droplet and activates the lipolysis Hidrolyzes of stored triacylglycerol to free fatty acids

62.21 53.63

Interconversion of glycogen, glucose, and trehalose in storage and mobilization of carbohydrates

20.64

Tret1 transporter comp15307_c0_seq1

14.33

Trehalose secretion to hemolymph

their function predicted yet. The most abundant cytochrome P450 in fat body was 304a1 (FPKM 132.46), and it could be related to hormone metabolism or breakdown of insecticides (Adams et al., 2000). In tubules, this cytochrome showed lower abundance (FPKM 2.89), whereas 346b2 showed the higher abundance (FPKM 172,58), however the function of this isoform was not described yet.

organism is high exhibiting a wide range of substrates (Scott, 1999). They are expressed in many insect tissues, such as: nervous system, fat body, Malpighian tubules, antenna, and gut. In insects, P450s may metabolize several compounds, such as juvenile hormone, ecdysteroids, pheromones and analogous, allelochemicals, and insecticides (Scott and Wen, 2001). Fig. 1 shows the transcripts inferred to be cytochromes P450 in the fat body and tubules of Z. morio. For comparison, the analysis of P450 expression patterns in embryos and larvae of D. melanogaster indicated the expression of 17 cytochromes in tubules and 9 in fat body of larvae, whereas Cyp4p2 and Cyp4s3 were exclusive of the fat body. Both tissues expressed the P450 12d1, 4e3, 6g1, 6w1, 314a1 e 4p3 (Chung et al., 2008). Cytochromes P450 found in our analysis are most likely involved in ecdysone synthesis (18a1, 306a1, 314a1, 315a1), fatty acid metabolism (4c1), insecticides, and others xenobiotics metabolism (6a2, 6bq11, 6bq2, 6bq4). It is important to emphasize that several P450s had not

4.4. Glutathione S-transferases Glutathione S-transferases (GST) catalyze the conjugation reaction of electrophilic substrates with thiol group of reduced glutathione, removing reactive oxygen species from cells, and metabolizing toxic compounds resulting in products more soluble to water and faster excretion (Sheehan et al., 2001; Li et al., 2009). Their common substrates are quinones, aldehydes, ketones, and esters. The presence of these enzymes is also related to resistance of organophosphate insecticides such as dichlorodiphenyltrichloroethane (DDT) in insects (Sheehan 100

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Guanosine triphosphate is the pteridin (red pigment) precursor and the enzyme GTP cyclohydrolase catalyzes the first reaction of this pathway. Then after some reactions (not well understood), the isoxanthopterin is produced by xanthine dehydrogenase, this enzyme also converts hipoxanthine to xanthine and then to uric acid in purine metabolism (Summers et al., 1982). Enzymes involved in the biosynthesis of these pigments are found in many tissues. Tryptophan oxygenase and xanthine dehydrogenase are present in the fat body and Malpighian tubules. Kynurenine 3-monooxygenase are found in tubules and kynurenine formamidase in the fat body and other tissues. These data are corroborated by our results because we found these enzymes in both the fat body and tubules of Z. morio larvae. However, in both tissues these enzymes exhibited low abundance. Tubules and fat body displayed transcripts similar to kynurenine aminotransferase, however, in tubules this enzyme exhibited higher abundance (FPKM 133.28) than in the fat body. This enzyme in tubules showed higher abundance in comparison with other enzymes related to brown eye pigment biosynthesis pathways. This result suggests that in Z. morio larvae most of the kynurenin formed is converted in kynurenic acid.

Fig. 1. Diagram representing the transcripts similar to cytochromes P450 found in fat body and Malpighian tubules of Z. morio larvae.

et al., 2001). There are six groups of cytosolic GSTs in insects: delta, epsilon, omega, sigma, theta, and zeta (Liu et al., 2014). Fat body of Z. morio showed all cited groups of GSTs and tubules did not exhibit only zeta GST. Delta and epsilon GSTs are implicated in the metabolism of allogenic chemicals; sigma is involved in some housekeeping-related roles and also insecticide metabolism (Liu et al., 2014). As cited above, one of the most abundant transcripts in tubules is a sigma GST (FPKM 3303.65), whereas in the fat body an epsilon GST was more abundant (FPKM 306.09). In T. molitor, an epsilon GST was specifically found in the fat body, and among the main GSTs which are expressed in tubules, one of them belongs to the sigma class (Liu et al., 2014).

4.7. AMP-CoA ligases in tubules and fat body AMP-CoA ligases, or adenylate-forming enzymes, catalyze the activation of carboxylic substrates by condensing them with ATP with release of pyrophosphate, usually followed by thioesterification of the carboxylic group to CoA (Viviani, 2002; Schmeiz and Naismith, 2009). These enzymes belong to an enzyme superfamily that play many roles, such as: fatty acids activation (acyl-CoA ligases), amino acids activation (aminoacyl-tRNA synthetases), bioluminescent reaction of Coleoptera species (luciferases), phenylpropanoid activation in plants (4-coumarate-CoA ligase), and xenobiotics metabolism (medium fatty-acid CoA synthetases) (Knights and Drogemuller, 2000; Stuible et al., 2000; Viviani, 2002; Schmeiz and Naismith, 2009). In mammals liver and kidneys, acyl-CoA synthetases enzymes are also involved in phase II of xenobiotics detoxification, and exhibit a wide range of substrates, such as benzoate, 4-chlorobenzoate, and hexanoate, besides fatty-acids (Knights and Drogemuller, 2000). Although beetle luciferases are well studied, their origin and evolution remains a mystery. Luciferase-like enzymes found in bioluminescent and non-bioluminescent Coleoptera are being used as models to understand the origin and evolution of bioluminescence in this insect group, and to understand the diversification of functions of ligases in insects (Viviani et al., 2009). Noteworthy, the transcriptional analysis of Z. morio larval tissues identified ~20 transcripts similar to 4-coumarate-CoA ligases in the Malpighian tubules and 13 transcripts in the fat body (~40% of identity). In plants, there are isoforms of 4-coumarate-CoA ligases (4CL) with different substrates. These enzymes catalyze the activation of cinnamic acid derivatives, such as 4-coumaric acid, caffeic acid, and ferulic acid, which participate in several reactions of phenylpropanoid metabolism (Stuible et al., 2000; Schneider et al., 2003). Table 6 shows the primary structure identity between transcripts similar to 4CLs (amino acids > 400) and the luciferase like-enzyme previously cloned from Z. morio tubules (Viviani et al., 2009). Noteworthy, the analysis performed alignments indicated that both tissues display ligases distinct from the luciferase-like enzyme previously cloned from the tubules (Viviani et al., 2009), exhibiting identity in the range of 21–92%. The absence of a gene product with 100% identity with the luciferase-like enzyme previously cloned in our analysis could be explained by the fact that we used distinct lineages of mealworms arising from different places (USA and Brazil). The fat body and tubules also express different isoforms of ligases and the abundance of these enzymes in both tissues was low (Table 6). Plants also express several isoforms of 4CLs in different tissues, and it is proposed that these isoforms could be specialized in activation of distinct cinamic substrates

4.5. Nitrogen metabolism Terrestrial insects excrete nitrogen from purine and amino acids metabolism mainly as uric acid (Klowden, 2007). The fat body is the main site for uric acid production and this compound often precipitates in the Malpighian tubules and gut (Nation, 2015). Enzymes that catalyze the last step in uric acid formation (xanthine dehydrogenase) and those associated with production of acid uric precursors, such as xanthine and hypoxanthine were found in both tissues. However, transcripts similar to xanthine dehydrogenase exhibited lower abundance in the fat body than in tubules. As previously cited, the purine metabolism was the pathway that showed the higher transcript number in the fat body (680 transcripts) and tubules (706 transcripts). This pathway carry out the formation of uric acid precursors from guanine and adenine metabolism. 4.6. Eye pigment biosynthesis The eye color in D. melanogaster is due to the presence of two types of pigments, ommochrome (brown) and pteridin (red). Ommochrome pigments are derived from tryptophan amino acid and their biosynthesis pathway involve some reactions catalyzed by different enzymes (Fig. A.2). The enzyme tryptophan oxygenase degrades the excess of tryptophan for excretion, storing it as kynurenine or 3-hydroxykynurenine, or converting it in xanthommatin (pigment). The main site to synthesis of kynurenine in larvae and developing adults is the fat body, whereas in adults the synthesis takes place in fat body and eyes. Therefore, the kynurenine formed in fat body is transported to Malpighian tubules where it is converted in 3-hydroxykynurenine (Summers et al., 1982). Kynurenine can be also be converted in kynurenic acid by kynurenine aminotransferase enzyme (Han et al., 2007). The tubules are involved in biosynthesis and storage precursors of pigments and also play an important role in excretion of these metabolites (Summers et al., 1982). 101

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Table 6 Alignment identity between transcripts similar to 4-coumarate-CoA ligases from analyzed tissues and luciferase like-enzyme previously cloned from Malpighian tubules of Z. morio. Malpighian tubules

Fat body

Transcript

FPKM

Amino acids residues

Identity with luciferase like-enzyme (%)

Transcript

FPKM

Amino acids residues

Identity with luciferase like-enzyme (%)

17,151 12,540 17,299 18,831 18,957 18,889 19,517 20,374 20,839 20,890 20,938 21,052

3.74 6.69 9.22 24.26 46.65 14.68 19.57 13.74 7.20 77.1 3.75 11.14

500 430 486 471 570 529 555 551 474 574 425 425

25 49 37 52 38 55 26 28 52 29 29 29

12,994 14,445 13,996 7728 15,990 15,351

3.66 32.65 43.46 4.06 13.69 4.41

523 529 528 555 581 406

92 50 26 22 23 21

relationship of the AMP-CoA ligases obtained in the fat body and Malpighian tubules of Z. morio with other enzymes of this superfamily (Fig. 2). We clearly identified a strong supported group that is formed by luciferase enzymes of Coleoptera species, as well as other related grouped formed by luciferase-like enzymes of Coleoptera and Diptera. Ligases from Z. morio are grouped close to 4CLs enzymes of Tribolium castaneum (Coleoptera) and Bombyx mori (Lepidoptera), and AMP-CoA ligase of Culex quinquefasciatus (Diptera). The relationships among 4CLs of Arabidopsis thaliana (Plantae) and Streptomyces (Bacteria), AMP-CoA ligases of C. quinquefasciatus and Aedes aegypti (Diptera), and 2-succinylbenzoate-CoA ligase are not completely defined. Furthermore, the distance of those proteins to the current beetle luciferases indicate that the carboxylic substrate specificity changed during the evolution and are completely distinct nowadays.

(Schneider et al., 2003). Transcriptional analysis of Phrixothrix hirtus (Phengodidae), a bioluminescent species, and the non-luminescent Chauliognathus opacus (Cantharidae) also showed the presence of 4CLs-like enzymes in lanterns (13 copies) and fat body (1 copy) of P. hirtus, and about 40 copies in the abdomen of C. opacus (Amaral et al., 2017a). Alignments of ligases from C. opacus and Z. morio fat body displayed identity in the range of 21–52% (data not shown). Therefore, these results may indicate that Coleoptera species express distinct isoforms of 4CLs-like ligases in different tissues. A transcript similar to a 4CLs-like enzyme from the tubules was amplified by RT-PCR (~600 pb), however, the amplification was weak. This corroborate the low abundance these enzymes in tubules (Table 6), and also explains their absence in the previous study of the limited transcriptional profile from Malpighian tubules cDNA library of Z. morio larvae (Silva et al., 2015). The absence of amplified products of 4CLs-like enzymes in the fat body also indicates the low abundance of these enzymes in the fat body (Table 6).

4.9. Multialignment of AMP-CoA ligases and carboxylic substrate inference Adenylate-forming enzymes share three conserved regions which are involved with adenylation and ATP ligation: motif I [SSG(S/ T)TGLPKG], motif II [GYGLTE], and motif III [LRTGD] (Viviani, 2002). Selected ligases found in our analysis show these motifs and also the motif [GEICIRG] (Fig. 3). This motif is conserved in 4-coumarate-CoA ligases (4CLs) in plants and it is suggested to be involved in the

4.8. Phylogenetic analysis of AMP-CoA ligases We performed a phylogenetic analysis using some available sequences on GenBank of AMP binding proteins to try to elucidate the

Fig. 2. Phylogenetic tree of AMP-CoA ligases based on Bayesian analysis. In red Coleoptera species: Photinus pyralis, Phrixothrix hirtus, Rhagophthalmus ohbai, Tenebrio castaneum, Tenebrio molitor, Zophobas morio. In blue Diptera species: Aedes aegypti, Arachnocampa luminosa, Culex quinquefasciatus, Drosophila melanogaster. In black other analyzed species: Arabidopsis thaliana (Plantae: Brassicales), Bombyx mori (Arthropoda: Lepidoptera), Escherichia coli (Bacteria: Enterobacteriales), Pseudomonas putida (Bacteria: Pseudomonadales), Streptomyces sp. (Bacteria: Actinomycetales). 102

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Fig. 3. Sequence multialignment of conserved motifs of adenylate-forming enzymes superfamily: MT ligase 17,151, ligase from Malpighian tubules of Z. morio; FB ligase 15,990, ligase from fat body of Z. morio; 4-coumarate-CoA ligase 2 of Arabidopsis thaliana (OAP07084.1); Proto Zop, luciferase like-enzyme from Malpighian tubules of Z. morio; Luc Ppy, luciferase of Photinus pyralis AB644228.1; Luc P. hirtus, luciferase of Phrixothrix hirtus FJ54529.1. In gray conserved motifs among adenylate-forming ligases (motifs I, II, III); yellow putative luciferin-binding site in luciferases; green conserved motif in 4-coumarate-CoA ligases. Residues that constitute SBP in At4CL2 isoform of A. thaliana are marked by an *.

Gly322, Ala323, Gly346, Gly348, Pro354, Val355, and Leu356 (Schneider et al., 2003). The multialignment of AMP-CoA ligases performed in our study shows the respective positions of the SBP amino acids residues in At4CL2 in comparison to ligases from Z. morio (Fig. 3). The cited model for At4CL2 isoform showed that SBP accessibility for monomethoxylated and dimethoxylated cinnamic acids derivatives is controlled mainly by size exclusion. The cinnamic acid conversion is mediated by the overall hydrophobicity of the SBP. The model also proposed that cinnamic acids derivatives are positioned in the SBP of At4CL2 in strictly oriented manner and the hydrogen bond between Asn-256 and 4-hydroxyl group of cinnamic acids derivatives stabilizing the orientation of the substrate within the SBP (Schneider et al., 2003). In the corresponding position, the ligases from Z. morio exhibited a threonine (T283), which also may stabilize the cinnamic acids

stabilization of the protein structure (Stuible et al., 2000). Furthermore, AMP-CoA ligases also show specific motifs carboxylic substrate binding. Plants 4CLs activate cinnamic acid derivatives (coumaric, caffeic, and ferulic acids), and exhibit different affinities to these substrates (Stuible et al., 2000; Schneider et al., 2003). Some studies showed regions related to substrates affinities for these enzymes in plants (Stuible et al., 2000; Schneider et al., 2003; Hu et al., 2010). Considering AMP-CoA ligases enzymes in tubules and fat body of Z. morio obtained in this study are similar to 4CLs enzymes of plants, it is possible to compare regions of substrate specificity between 4CL and Z. morio 4CL like-enzymes to infer their possible substrates. In A. thaliana 4CL isoform (At4CL2) a signature motif of 12 amino acids, called substrate binding pocket (SBP), was identified. Amino acids residues of the SBP are Ile252, Tyr253, Asn256, Met293, Lys320, 103

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derivatives or similar compounds by hydrogen bond. In At4CL2 mutants, the presence of small amino acids at either position 293 or 320 (wild type Met293 and Lys320) is important for ferulic acid activation (Schneider et al., 2003). Therefore, the presence of Phe320 and Val347 in the ligases from Z. morio may, corresponding to positions 293 and 320 of At4CL2 isoform, prevent ferulic acid activation by size exclusion. Some 4CLs isoforms have a deletion mutation at Val338 that allow them to bind to the larger sinapic acid (Hu et al., 2010). The deletion position corresponds to Met383 in Z. morio ligases. This amino acid exhibits a long side chain that could prevent the ligation of larger carboxylic substrates, such as sinapic acid. Therefore, it is possible that in Z. morio such 4CL-like ligases are capable to activate smaller cinnamic acids derivatives, such as coumaric and caffeic acids. The roles of luciferase like-enzymes and other AMP-CoA ligases in tubules and fat body remain unknown, however, some evidences suggest that luciferase like-enzyme could be involved with the detoxification/excretion of carboxylic acids in Malpighian tubules (Prado et al., 2016). Cinnamic acids derivatives (coumaric, caffeic, ferulic) are found in wheat bran (Onyeneho and Hettiarachchy, 1992), thus 4-coumarateCoA like enzymes of Z. morio tubules could be involved in the detoxification of phenolic acids obtained from diet. Alternatively, these enzymes could be involved in the activation of acids that are structurally similar to phenylpropanoid acids metabolism in plants during pigment biosynthesis in insects.

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5. Conclusions Although classical physiological studies showed that Malpighian tubules and fat body play distinct physiologic roles in insects, our analysis indicated that they share important functions such as eye pigment synthesis, nitrogen metabolism to excretion, immunity, and detoxification. The results also confirm that tubules and fat body are versatile tissues in the coleopteran larvae. The presence of 4-coumarateCoA ligase-like enzymes indicate their possible involvement in important physiological roles for tubules and fat body physiology, like the activation and detoxification of phenolic acids (cinnamic acids derivatives) obtained from diet, or the activation of acids that are structurally similar to cinnamic acids derivatives during insect pigment biosynthesis. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbd.2018.11.007. Acknowledgements Thanks to Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP grant numbers: 2010/05426-8, 2013/09594-0, 2012/ 25378-3); and Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq grant number: 401867/2016-1) and Conselho de Aperfeiçoamento de Ensino Superior (CAPES) for financial support. Thanks to Nilson Silva for statistical support. References Abascal, F., Zardoya, R., Posada, D., 2005. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105. Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., 2000. The genome sequence of Drosophila melanogaster. Science 5461, 2185–2195. Amaral, D.T., Silva, J.R., Viviani, V.R., 2017a. Transcriptional comparison of the photogenic and non-photogenic tissues of Phrixothrix hirtus (Coleoptera: Phengodidae) and non-luminescent Chauliognathus opacus (Coleoptera: Cantharidae) give insights on the origin of lanterns in railroad worms. Gene Rep. 7, 78–86. Amaral, D.T., Silva, J.R., Viviani, V.R., 2017b. Transcriptomes from the photogenic and non-photogenetic tissues and life stages of the Aspisoma lineatum firefly (Coleoptera: Lampyridae): implications for the evolutionary origins of bioluminescence and its associated light organs. Gene Rep. 8, 150–159. Andrews, S., 2010. FastQC: a quality control tool for high throughput sequence data. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation.

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