Exploring the contribution of the salivary gland and midgut to digestion in the swede midge (Contarinia nasturtii) through a genomics-guided approach.

The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and ß-fructofuranosidases (invertases) were identified. Genes encoding ß-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge.

Fatty Acid Desaturase Bmdesat5, Suppressed in the Salivary Glands by Domestication, is Involved in Regulation of Food Intake in Silkworms.

Understanding the evolutionary genetics of food intake regulation in domesticated animals has relevance to evolutionary biology, animal improvement, and obesity treatment. Here, we observed that the fatty acid desaturase gene (Bmdesat5), which regulates food intake, is suppressed in domesticated silkworms, but expressed in the salivary glands of the wild silkworm Bombyx mandarina. The content of its catalytic product, cis-vaccenic acid, was related to the expression levels of Bmdesat5 in the salivary glands of domesticated and wild silkworm strains. These two strains also showed significant differences in food intake. Using orally administering cis-vaccenic acid and transgenic-mediated overexpression, we verified that cis-vaccenic acid functions as a satiation signal, regulating food intake and growth in silkworms. Selection analysis showed that Bmdesat5 experienced selection, especially in the potential promoter, 5'-untranslated, and intron regions. This study highlights the importance of the decrement of satiety in silkworm domestication and provides new insights into the potential involvement of salivary glands in the regulation of satiety in animals, by acting as a supplement to gut-brain nutrient signaling.

Trypanosoma cruzi infection modulates secreted phospholipase A2 expression in the salivary glands of Rhodnius prolixus.

Phospholipases A2 (PLA2) comprise a superfamily of enzymes that specifically catalyze hydrolysis of the ester bond at the sn-2 position of glycerophospholipids, generating lysophospholipids and fatty acids. In Rhodnius prolixus, one of the main vectors of the Chagas's disease etiologic agent Trypanosoma cruzi, it was previously shown that lysophosphatidylcholine, a bioactive lipid, found in the insect's saliva, contributes to the inhibition of platelet aggregation, and increases the production of nitric oxide, an important vasodilator. Due to its role in potentially generating LPC, here we studied the PLA2 present in the salivary glands of R. prolixus. PLA2 activity is approximately 100 times greater in the epithelium than in the contents of salivary glands. Our study reveals the role of the RpPLA2XIIA gene in the insect feeding performance and in the fatty acids composition of phospholipids extracted from the salivary glands. Knockdown of RpPLA2XIIA significantly altered the relative amounts of palmitic, palmitoleic, oleic and linoleic acids. A short-term decrease in the expression of RpPLA2III and RpPLA2XIIA in the salivary glands of R. prolixus was evident on the third day after infection by T. cruzi. Taken together, our results contribute to the understanding of the role of PLA2 in the salivary glands of hematophagous insects and show that the parasite is capable of modulating even tissues that are not colonized by it.

An inhibitor of leukotriene-A4 hydrolase from bat salivary glands facilitates virus infection.

SignificanceAn immunosuppressant protein (MTX), which facilitates virus infection by inhibiting leukotriene A4 hydrolase (LTA4H) to produce the lipid chemoattractant leukotriene B4 (LTB4), was identified and characterized from the submandibular salivary glands of the bat Myotis pilosus. To the best of our knowledge, this is a report of an endogenous LTA4H inhibitor in animals. MTX was highly concentrated in the bat salivary glands, suggesting a mechanism for the generation of immunological privilege and immune tolerance and providing evidence of viral shedding through oral secretions. Moreover, given that the immunosuppressant MTX selectively inhibited the proinflammatory activity of LTA4H, without affecting its antiinflammatory activity, MTX might be a potential candidate for the development of antiinflammatory drugs by targeting the LTA4-LTA4H-LTB4 inflammatory axis.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.

Expression Profiles of Digestive Genes in the Gut and Salivary Glands of Tarnished Plant Bug (Hemiptera: Miridae).

Host plant preference of agricultural pests may shift throughout the growing season, allowing the pests to persist on wild hosts when crops are not available. Lygus Hahn (Hemiptera: Miridae) bugs are severe pests of cotton during flowering and fruiting stages, but can persist on alternative crops, or on weed species. Diversity of digestive enzymes produced by salivary glands and gut tissues play a pivotal role in an organism's ability to utilize various food sources. Polyphagous insects produce an array of enzymes that can process carbohydrates, lipids, and proteins. In this study, the digestive enzyme repertoire of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by high-throughput sequencing followed by cDNA cloning and sequencing. This study identified 87 digestive genes, including 30 polygalacturonases (PG), one ß-galactosidase, three α-glucosidases, six ß-glucosidases, 28 trypsin-like proteases, three serine proteases, one apyrase-like protease, one cysteine protease, 12 lipases, and two transcripts with low similarity to a xylanase A-like genes. RNA-Seq expression profiles of these digestive genes in adult tarnished plant bugs revealed that 57 and 12 genes were differentially expressed in the salivary gland and gut (≥5-fold, P ≤ 0.01), respectively. All polygalacturonase genes, most proteases, and two xylanase-like genes were differentially expressed in salivary glands, while most of the carbohydrate and lipid processing enzymes were differentially expressed in the gut. Seven of the proteases (KF208689, KF208697, KF208698, KF208699, KF208700, KF208701, and KF208702) were not detected in either the gut or salivary glands.

Choline Acetyltransferase Induces the Functional Regeneration of the Salivary Gland in Aging SAMP1/Kl -/- Mice.

Salivary gland dysfunction induces salivary flow reduction and a dry mouth, and commonly involves oral dysfunction, tooth structure deterioration, and infection through reduced salivation. This study aimed to investigate the impact of aging on the salivary gland by a metabolomics approach in an extensive aging mouse model, SAMP1/Klotho -/- mice. We found that the salivary secretion of SAMP1/Klotho -/- mice was dramatically decreased compared with that of SAMP1/Klotho WT (+/+) mice. Metabolomics profiling analysis showed that the level of acetylcholine was significantly decreased in SAMP1/Klotho -/- mice, although the corresponding levels of acetylcholine precursors, acetyl-CoA and choline, increased. Interestingly, the mRNA and protein expression of choline acetyltransferase (ChAT), which is responsible for catalyzing acetylcholine synthesis, was significantly decreased in SAMP1/Klotho -/- mice. The overexpression of ChAT induced the expression of salivary gland functional markers (α-amylase, ZO-1, and Aqua5) in primary cultured salivary gland cells from SAMP1/Klotho +/+ and -/- mice. In an in vivo study, adeno-associated virus (AAV)-ChAT transduction significantly increased saliva secretion compared with the control in SAMP1/Klotho -/- mice. These results suggest that the dysfunction in acetylcholine biosynthesis induced by ChAT reduction may cause impaired salivary gland function.

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem.

Recent studies have reported that aphids facilitate their colonization of host plants by secreting salivary proteins into host tissues during their initial probing and feeding. Some of these salivary proteins elicit plant defenses, but the molecular and biochemical mechanisms that underlie the activation of phloem-localized resistance remain poorly understood. The aphid Myzus persicae, which is a generalized phloem-sucking pest, encompasses a number of lineages that are associated with and adapted to specific host plant species. The current study found that a cysteine protease Cathepsin B3 (CathB3), and the associated gene CathB3, was upregulated in the salivary glands and saliva of aphids from a non-tobacco-adapted (NTA) aphid lineage, when compared to those of a tobacco-adapted lineage. Furthermore, the knockdown of CathB3 improved the performance of NTA lineages on tobacco, and the propeptide domain of CathB3 was found to bind to tobacco cytoplasmic kinase ENHANCED DISEASE RESISTANCE 1-like (EDR1-like), which triggers the accumulation of reactive oxygen species in tobacco phloem, thereby suppressing both phloem feeding and colonization by NTA lineages. These findings reveal a novel function for a cathepsin-type protease in aphid saliva that elicits effective host plant defenses and warranted the theory of host specialization for generalist aphids.

Extraoral digestion: outsourcing the role of the hemipteran midgut.

Extraoral digestion allows for breakdown of dietary components before they reach the midgut for final enzymatic degradation and absorption. In the Hemiptera, this is achieved by the secretion of enzyme-rich fluids from the salivary gland, with the combination of protein and mRNA from these tissues termed the sialome. Separate channels within the hemipteran stylets allow for secretion of saliva and ingestion of predigested material in a non-reflux mechanism. Both feeding mode and diet type influence the composition of the hemipteran sialome, as illustrated by 1) differences in protease abundance between hematophagous and predatory heteropteran sialomes, 2) diet specific aminopeptidase-N genes among aphid biotypes, and 3) adaptation-induced sialome variation in related cicada populations. Despite challenges associated with incomplete genome annotation, -omics analysis of the sialomes of diverse hemipteran species will enhance understanding of both sialome function and the evolution of extraoral digestion within the order.

CRISPR/Cas9-Mediated Integration of Large Transgene into Pig CEP112 Locus.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating tool that can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knockout, the gene knock-in (KI) efficiency mediated by homology-directed repair remains low, especially for large fragment integration. In this study, we established an efficient method for the CRISPR/Cas9-mediated integration of large transgene cassette, which carries salivary gland-expressed multiple digestion enzymes (≈ 20 kbp) in CEP112 locus in pig fetal fibroblasts (PFFs). Our results showed that using an optimal homology donor with a short and a long arm yielded the best CRISPR/Cas9-mediated KI efficiency in CEP112 locus, and the targeting efficiency in CEP112 locus was higher than in ROSA26 locus. The CEP112 KI cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that KI pig (705) successfully expressed three microbial enzymes (ß-glucanase, xylanase, and phytase) in salivary gland. This finding suggested that the CEP112 locus supports exogenous gene expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus in pigs by using our optimal homology arm system and established a modified pig model for foreign digestion enzyme expression in the saliva.

ROCK inhibitor increases proacinar cells in adult salivary gland organoids.

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies.

Proteases and nucleases across midgut tissues of Nezara viridula (Hemiptera:Pentatomidae) display distinct activity profiles that are conserved through life stages.

The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.

Malaria load affects the activity of mosquito salivary apyrase.

Mosquitoes infected by sporozoites, the infectious stage of malaria, bite more frequently than uninfected mosquitoes. One of the mechanisms underlying this behavioural change appears to be that the sporozoites decrease the activity of apyrase, an ADP-degrading enzyme that helps the mosquitoes to locate blood. Using the parasite Plasmodium berghei and the mosquito Anopheles gambiae, we confirmed that sporozoite infection alters the host-seeking behaviour of mosquitoes by making them more likely to refeed after a first blood meal, and that apyrase activity is one of the mechanisms of the increased biting persistence and motivation of infectious mosquitoes. We further showed that apyrase activity decreases as the sporozoite load increases, and that mosquitoes with lower apyrase activity take up less blood, making it more likely that they would return to top up their blood meal. Finally, by comparing full-sib families of mosquitoes, we showed that there was genetic variation for apyrase activity, but not for the resistance of parasites to be manipulated. Our results give new insights in understanding how malaria parasites change their hosts to affect their own transmission.

The C-Terminal Domain of RNA Polymerase II Is a Multivalent Targeting Sequence that Supports Drosophila Development with Only Consensus Heptads.

The C-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. On the contrary, we show that a truncated CTD composed solely of YSPTSPS repeats supports Drosophila viability but that a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci separate from the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

Herbivorous Caterpillars Can Utilize Three Mechanisms to Alter Green Leaf Volatile Emission.

Green plants emit green leaf volatiles (GLVs) as a general damage response. These compounds act as signals for the emitter plant, neighboring plants, and even for insects in the ecosystem. However, when oral secretions from certain caterpillars are applied to wounded leaves, GLV emissions are significantly decreased or modified. We examined four caterpillar species representing two lepidopteran families for their capacity to decrease GLV emissions from Zea mays leaf tissue. We also investigated the source of the GLV modifying components in the alimentary tract of the various caterpillars. In Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), we found three distinct mechanisms to modify GLV emission: a heat-stable compound in the gut, a heat-labile enzyme in salivary gland homogenate (previously described in Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and an isomerase in the salivary gland homogenate, which catalyzes the conversion of (Z)-3-hexenal to (E)-2-hexenal (previously described in M. sexta). These mechanisms employed by caterpillars to suppress or modify GLV emission suggest a counteraction against the induced indirect volatile defenses of a plant and provides further insights into the ecological functions of GLVs.

Tissue-specific transcription of proteases and nucleases across the accessory salivary gland, principal salivary gland and gut of Nezara viridula.

The phytophagous stink bug, Nezara viridula (L.) infests multiple plant species and impacts agricultural production worldwide. We analyzed the transcriptomes of N. viridula accessory salivary gland (ASG), principal salivary gland (PSG) and gut, with a focus on putative digestive proteases and nucleases that present a primary obstacle for the stability of protein- or nucleic acid-based stink bug control approaches. We performed high throughput Illumina sequencing followed by de novo transcriptome assemblies. We identified the sequences of 141 unique proteases and 134 nucleases from the N. viridula transcriptomes. Analysis of relative transcript abundance in conjunction with previously reported proteome data (Lomate and Bonning, 2016) supports high levels of serine protease expression in the salivary glands and high cysteine protease expression in the gut. Specifically, trypsin and chymotrypsin transcripts were abundant in the PSG, and cathepsin L-like cysteine protease transcripts were abundant in the gut. Nuclease transcript levels were generally lower than those of the proteases, the exception being abundant transcripts of ribonuclease-C20 in the PSG. The abundance of chymotrypsin, trypsin, and some carboxypeptidase transcripts suggests a significant role for the PSG in production of digestive enzymes. This result is at odds with the premise that the ASG produces watery saliva, which is high in enzymatic activity, while the PSG produces only sheath saliva. We have generated a comprehensive transcriptome sequence dataset from the digestive organs of N. viridula, identified major protease and nuclease genes and confirmed expression of the most abundant enzymes thereby providing greater insight into the digestive physiology of N. viridula.

Molecular and biochemical characterization of the bed bug salivary gland cholinesterase as an acetylcholine-sequestering enzyme.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this study, we investigated the molecular forms, tissue distribution patterns and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was confirmed by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of hydrolysis of acetylcholine (ACh) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. ClSChE binding to ACh was not clearly resolved in the binding assay format used in this study, probably due to the weak but detectable ACh-hydrolytic activity of ClSChE. Nevertheless, kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions virtually as an ACh-sequestering protein by having a very strong affinity to ACh but an extremely long turnover time. Given that ACh regulates a wide variety of host physiologies, we discuss the tentative roles of ClSChE in blood vessel constriction and itch/pain regulation in the host.

Novel transgenic pigs with enhanced growth and reduced environmental impact.

In pig production, inefficient feed digestion causes excessive nutrients such as phosphorus and nitrogen to be released to the environment. To address the issue of environmental emissions, we established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, ß-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphorus outputs in the transgenic pigs were reduced by 23.2-45.8%, and growth rate improved by 23.0% (gilts) and 24.4% (boars) compared with that of age-matched wild-type littermates under the same dietary treatment. The transgenic pigs showed an 11.5-14.5% improvement in feed conversion rate compared with the wild-type pigs. These findings indicate that the transgenic pigs are promising resources for improving feed efficiency and reducing environmental impact.

Marine Leech Anticoagulant Diversity and Evolution.

Leeches (Annelida: Hirudinea) possess powerful salivary anticoagulants and, accordingly, are frequently employed in modern, authoritative medicine. Members of the almost exclusively marine family Piscicolidae account for 20% of leech species diversity, and they feed on host groups (e.g., sharks) not encountered by their freshwater and terrestrial counterparts. Moreover, some species of Ozobranchidae feed on endangered marine turtles and have been implicated as potential vectors for the tumor-associated turtle herpesvirus. In spite of their ecological importance and unique host associations, there is a distinct paucity of data regarding the salivary transcriptomes of either of these families. Using next-generation sequencing, we profiled transcribed, putative anticoagulants and other salivary bioactive compounds that have previously been linked to blood feeding from 7 piscicolid species (3 elasmobranch feeders; 4 non-cartilaginous fish feeders) and 1 ozobranchid species (2 samples). In total, 149 putative anticoagulants and bioactive loci were discovered in varying constellations throughout the different samples. The putative anticoagulants showed a broad spectrum of described antagonistic pathways, such as inhibition of factor Xa and platelet aggregation, which likely have similar bioactive roles in marine fish and turtles. A transcript with homology to ohanin, originally isolated from king cobras, was found in Cystobranchus vividus but is otherwise unknown from leeches. Estimation of selection pressures for the putative anticoagulants recovered evidence for both positive and purifying selection along several isolated branches in the gene trees, and positive selection was also estimated for a few select codons in a variety of marine species. Similarly, phylogenetic analyses of the amino acid sequences for several anticoagulants indicated divergent evolution.

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg2+, Co2+, Cu2+ and Zn2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses.