UNIVERSIDAD DE LA FRONTERA

Facultad de Ingeniería y Ciencias

Programa de Doctorado en Ciencias de Recursos Naturales

EFFECTS OF PROTEASES INHIBITORS ON THE DIGESTIVE SYSTEM, FEEDING AND DEVELOPMENT OF Aegorhinus superciliosus RASPBERRY WEEVIL (GUÉRIN-MÉNEVILLE, 1830) (COLEOPTERA: CURCULIONIDAE)

DOCTORAL THESIS IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF SCIENCES IN NATURAL RESOURCES

VIVIAN ELIETTE MEDEL MEZA TEMUCO – CHILE 2020

EFFECTS OF PROTEASES INHIBITORS ON THE DIGESTIVE SYSTEM, FEEDING AND DEVELOPMENT OF Aegorhinus superciliosus RASPBERRY WEEVIL (GUÉRIN-MÉNEVILLE, 1830) (COLEOPTERA: CURCULIONIDAE)

Esta tesis fue realizada bajo la supervisión del Doctor Andrés Quiroz Cortez, perteneciente al

Departamento de Ciencias Químicas y Recursos Naturales de la Universidad de La Frontera y es

presentada para su revisión por los miembros de la comisión examinadora.

Dr. Andrés Quiroz Cortez Dr. Andrés Quiroz C. Director del Programa de Doctorado en Ciencias de Recursos Naturales

Dra. Marysol Alvear Z.

Dra. Mónica Rubilar Director Académico de Postgrado Universidad de La Frontera Dr. Luis Salazar

Dr. Eduardo Fuentes

Dr. Cristian Figueroa

A la memoria de mi Padre Miguel Ángel Medel Ramos, quien me enseñó a mirar la vida de frente, a enfrentar los problemas con entereza y a levantarme cada vez que caía…porque La vida se rinde al valeroso y se ensaña con el que se acobarda….

Agradecimientos

Este largo camino recorrido estuvo lleno de dificultades, tantas que ya perdí la cuenta, pero lo que no olvidaré nunca, es que a pesar de todo Dios me regaló la vida por segunda vez y ese día me aferré a esta vida que casi me fue arrebatada y aprendí a disfrutar y vivir intensamente cada minuto de ella. Dar las gracias a veces no es suficiente, pues tendría que vivir tres vidas para pagarles de alguna manera a todos quienes de una u otra forma me ayudaron y estuvieron conmigo en todo momento.

Agradezco a DIOS, que en su infinita bondad me permitió llegar a vivir este mágico momento, pues sin duda él tuvo mucho que ver con este logro. A mis padres Miguel Ángel y Mariana, quienes una vez más me han demostrado su amor infinito y su apoyo incondicional, pues se transformaron en mis ojos, mis manos y mis piernas en todo este largo camino, y sin ellos esto no habría sido posible ni siquiera en sueños. A mi Padre, Miguel Ángel quien me dejó demasiado pronto, pero quien mientras estuvo conmigo me profesó un amor profundo e infinito, tanto así que llegó muchas veces a aprender lo que no imaginó para ayudarme en mis ensayos.

Hoy que ya no lo tengo conmigo le dedico este trabajo que no estuvo exento de sin sabores y lágrimas, de dolor y angustia, pero que, según él, algún día daría sus frutos.

Doy las gracias a mis tres hijos, a Vivian Eliette, por quien siento admiración y orgullo porque es una hija maravillosa, quien en silencio aceptó casi una madre virtual, pero a pesar de eso me apoyó y jamás me criticó. A Diego Alfonso y Paloma Patricia, mis compañeros en la búsqueda de mis insectos y quienes no entendían muchas veces que su madre estuviera ausente o dejara de jugar con ellos por dedicarle tiempo al doctorado, pero que sin ninguna crítica me demostraban su amor con palabras, abrazos y toda su ternura. A mi hermana Edith Mariana

i quien a pesar de mi gran falta que casi le cuesta la vida, ha estado conmigo en todo momento compartiendo mis alegrías y también mis penas. A mis sobrinos Jorge Andrés y Miguel Ángel

Alonso mis otros tesoros a quienes agradezco todo el amor que me dan.

A mi esposo Ramón Eduardo quien me incentivó a realizar el doctorado, pero quien nunca entendió que para una mujer con esposo e hijos es una prueba dura y difícil de lograr, quien criticó muchas veces mi ausencia y no supo entenderme, doy las gracias porque a pesar de eso fue mi compañero en la colecta de mis insectos y me hizo entender que soy capaz de hacer cosas que no creía posibles.

A quienes llegaron a mi vida sin pensarlo, tal vez sin quererlo y trabajaron conmigo de manera desinteresada llegando a convertirse en personas muy queridas y a quienes debo momentos maravillosos y todos los conocimientos obtenidos en estos años, a Don Eduardo Gutiérrez, quien fue el primero en tener paciencia y voluntad para apoyarme con la bioquímica y quien me incentivó a creer en mis capacidades, al Dr. Héctor Rocha, un hombre bondadoso, quien dedico muchos fines de semana para trabajar conmigo, quien me enseñó que la bioquímica no es una tortura sino un reto, quien creyó en mí y me apoyó en todo este largo trabajo y por quien siento cariño y admiración. A don Benedicto Molina, parte importante de esta tesis, quien me abrió las puertas del laboratorio de histología, me permitió realizar las muestras histológicas, quien me apoya incondicionalmente hasta hoy y a quien aprendí a querer como a un amigo. A don Jorge Seguel, quien con paciencia y su experiencia me ayudó en la realización de las muestras histológicas, gracias por su tiempo y sus enseñanzas.

Al Dr. Ramón Rebolledo Ranz, quien fue gestor intelectual de gran parte de esta tesis, quien

ii facilitó su laboratorio y todos los implementos necesarios para realizar mis ensayos, gracias a su apoyo este trabajo pudo ser concretado. A mi Tutor el Dr. Andrés Quiroz Cortez, quien por travesuras del destino me recibió como tesista y en los momentos más duros del doctorado me apoyó incondicionalmente, creyó en mí y me permitió lograr mis objetivos.

Gracias profundamente a la Dra. Marysol Alvear Zamora, a quien conozco desde mis años de estudiante de pre grado y quien ha sido una persona especial para mí, quien me ha aconsejado, enseñado y también entregado su cariño. Gracias por sus consejos y por ser esos brazos tiernos que se necesitan cuando las cosas salen mal. A la Sra. María Eugenia Osses, quien fue mi concejera más allá del inglés de mis textos, su paciencia y dedicación la hicieron ganarse mi corazón y mi cariño.

A mis amigos quienes estuvieron conmigo en las malas y en las buenas, aún en la distancia y a quienes no les faltó el cariño, el abrazo o las palabras de ánimo fundamentales para que yo pudiera seguir adelante. A Henry Villagra, un hombre noble y sencillo, quien sin pensarlo se convirtió en mi amigo, en compañero de muchas vivencias, quien ha estado ahí siempre para solucionar mis problemas, gracias por tu maravillosa amistad, que en estos años ha sido fundamental, a Neftalí Faúndez un hombre bondadoso y paciente, quien me entregó su amistad y sus conocimientos… Gracias porque sin ti…esto no habría sido posible. A mis “amigas locas deschavetadas”, Pilar Vera, a pesar de que partiste antes que concluyera este trabajo, desde ese lugar especial sé que estas celebrando por esto…estarás en mi corazón y en mi alma por siempre.. y a Paulina Cabrera…quien me torturó constantemente para que pudiera terminar este trabajo…Gracias a ambas porque me enseñaron a creer en mí, a valorarme como profesional, como ser humano y me engrandecieron con su amistad.

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Un agradecimiento especial para todas aquellas personas anónimas de la Comisión Nacional

Científica y Tecnológica (CONICYT) del Gobierno de Chile, quienes me otorgaron la Beca de doctorado y la beca de término de tesis, sin las cuales este sueño no podría haberse hecho realidad.

A todas aquellas personas que aparecieron en mi camino y me brindaron su sonrisa…

A todas aquellas personas que en la distancia tenían sentimientos positivos para mí…

A todos aquellos seres generosos que me entregaron una palabra de ánimo cuando estaba desahuciada…

A todos quienes ayudaron a levantarme cuando caí…

A todos esos seres maravillosos que pudiera olvidar, y que en algún momento se cruzaron en mi camino y caminaron junto a mi….

Finalmente expreso mi gratitud a todos aquellos que simplemente estuvieron conmigo, cerca, lejos, aquí o allá y que siempre me brindaron su apoyo en silencio…

Y por último sólo me queda dar GRACIAS A LA VIDA……..

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Summary

Insect pests are one of the most important biotic factors affecting the agriculture. They are responsible for the severe reduction in crop yields, besides the use of pesticides that affect beneficial insects such as bees and contaminate waterways and environment. Based on their feeding behavior, insect pests are classified into two types, specialized feeders and polyphagous insects. Insects need an adequate source of protein in order to grow and maintain themselves. In this scenario, proteases are very important enzymes in the alimentary system of insects, breaking peptide bonds in the proteinous insect foods to release the amino acids that are then absorbed by epithelial cells of the insect midgut. Proteolysis plays an important role in insect physiology and food digestion, facilitated by serine, cysteine, aspartic and metalloproteases.

Protease inhibitors are proteins or small polypeptides that may interfere with food digestion in the insect gut. These inhibitors can bind with dominant digestive proteases of insects feeding on the host plants, impairing their digestion and retarding growth and development in some insect species.

Aegorhinus superciliosus Guérin-Méneville, 1830 (Coleoptera: Curculionidae) raspberry weevil is a native insect of the Southern Cone of America (Chile and Argentina), whose larvae have powerful jaws with which it cuts and grinds roots and rootlets of plants, finally piercing the taproot near the neck, where it builds a pupal chamber. This larval activity causes a detriment to the plant, eventually resulting in its death, whereby is considered a pest of economical importance in southern Chile and the most important pest of blueberry (Vaccinium corymbosum).

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This crop ranks as one of the most consumed berries for their high antioxidant and beneficial health properties. Farmers invest considerable resources to control it, which consists mainly of the use of chemicals.

In this investigation, the morphological and histological study of the digestive system of Aegorhinus superciliosus (Coleoptera: Curculionidae) was carried out, to assess whether protease inhibitors cause destruction of cell tissue, in addition the presence and types of proteases in the digestive tract were determined and the negative effect of these inhibitors on proteases and therefore on the feeding and development of the adult insect was evaluated. This species has a characteristic digestive system for most insects, being similar in males and females, but longer in females, reaching measuring twice the size of the insect. The digestive system is a long tube of variable diameter, extending from the pharynx to the anus and is divided into three regions: foregut (stomodeum), formed by the pharynx, esophagus and proventriculus ending called "cardiac" valve, the midgut

(mesenteron) divided into anterior and posterior ventricle, the latter having many gastric caeca and hindgut (proctodeum) that starts at the pyloric valve and proceeds to the ileum, colon, rectum, and leading to anus. This study revealed the presence of six Malpighian tubes that start in the pylorus and extend into its rectum.

Histologically, it presents an epithelial tissue similar to that described for other species and externally a muscle tissue, but there are no differences between males and females. However, it presents features that are absent in other Curculionides and Coleopters such as absence of crop, location and number of the gastric caeca, the presence of peritrofic membrane and absence of rectal pads.

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Gut extracts of adult were analyzed using different specific peptide substrates and proteases inhibitors. Trypsin and chymotrypsin were characterized at different pHs, and Michaelis- Menten constant for both was determined. Furthermore, the effect of specific inhibitors, in vitro assays, and the effect of soluble proteins extracted from vegetable sources on the proteolytic activity of trypsin and chymotrypsin were evaluated, using BApNA and SAAPFpNA as specific substrates, respectively.

The results indicated that pH 8.0 is the optimal of total proteolytic activity, suggesting that proteases present in the gut of the insect would be principally serine proteases. Using specific substrates, it was determined that trypsin and chymotrypsin have an optimal pH activity at 8.0 and

11.0, respectively. Kinetic studies showed a Km value of 0.28 ± 0.07 mM for trypsin and a Km value of 0.38 ± 0,021 mM for chymotrypsin. Enzyme inhibition assays in vitro using PMSF and

SBTI as inhibitors in concentrations of 0.01, 0.1 and 0.2 mM were performed showing percentages of inhibition between 0 and 16% for PMSF, between 67 and 76% for SBTI inhibitor; whereas peel soluble proteins obtained from potatoes (Yagana variety) caused 50% inhibition of enzymatic activity for the highest tested concentration (10 mg / mL).

In vivo effects of three commercial inhibitors (PMSF, TPCK and SBTI) were evaluated spraying them on the leaves of Rubus ulmifolius for five days before feeding insects, to identify their effectiveness, selectivity and sensitivity on digestive proteases of the adult. The results indicated that PMSF and TPCK exert an antifeedant effect on adult raspberr y weevil, likewise SBTI inhibitor exerts an insecticidal effect producing 100% mortality of individuals in the highest tested concentration, and PMSF causes 10% mortality the same as TPCK. Whereby, SBTI was the most effective inhibitor compared with the other two products applied. On the other hand, mortality

vii was higher in females than in males in the three doses tested.

The first step for discovering protease inhibitors is the identification and biochemical characterization of digestive proteases in the insect gut. Characterization and identification of the type of digestive proteases is fundamental for studying nutritional physiology and degradation or activation of toxin protein. Therefore, the use of protease inhibitors may be an effective alternative control of A. superciliosus, so future trials need to be conducted in the field to verify its feasibility as insecticide.

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TABLE OF CONTENTS

Agradecimientos i Summary v Table of contents ix

CHAPTER 1 General Introduction 1

1.1. General introduction 2

1.2. Hypotheses 15

1.3. General objectives 15

1.4. Specific objectives 15

CHAPTER 2 16

Morphological and histological characterization of the digestive system of

Aegorhinus superciliosus raspberry weevil (Coleoptera: Curculionidae).

Abstract 18

2.1. Introduction 19

2.2. Materials and methods 20

2.2.1. Biological material 20 2.2.2. Study place 20 2.2.3. Dissection 20 2.2.4. Histological preparation of the samples 21 2.3. Results 21

2.3.1. Morphological Description 21

2.3.1.1. Foregut 22 2.3.1.2. Midgut 23

2.3.1.3. Hindgut 23

2.3.2. Histological Description 25 2.3.2.1. Foregut 25 2.3.2.2. Midgut 26

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2.3.2.2.1. Anterior ventricle 26 2.3.2.2.2. Posterior ventricle 27 2.3.2.2.2.1. Gastric caeca 27 2.3.2.3 Hindgut 28 2.3.2.3.1. Ileum 28 2.3.2.3.2. Colon 29 2.3.2.3.3. Rectum 29 2.4. Discussion 31 2.5. Conclusions 35 2.6. Acknowledgements 35

CHAPTER 3 36

Effects of protease inhibitors on the digestive system, feeding and development of Aegorhinus superciliosus raspberry weevil (coleoptera: curculionidae)

Abstract 5 38

3.1. Introduction 540

3.2. Materials and methods 6 48

3.2.1. Adult insect collection 6 48

3.2.2. Plant material 6 48

3.2.3. In vivo assays 6 49

3.2.3.1. Insect mortality and antifeedant effect determination 6 49

3.2.4. Dissection 50

3.2.5. Histological preparation of samples 51

3.2.6. Experimental design 52

3.2.7. Data analysis 53

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3.3. Results and discussion 53

3.3.1. Effect of protease inhibitors on the tissue of digestive system 53

3.3.2. In vivo effects of protease inhibitors 59

3.3.2.1. Antifeedant effect of protease inhibitors 60

3.3.2.2. Insecticidal effect of protease inhibitors 61

3.4. Conclusions 64

3.5. Acknowledgements 66

CHAPTER 4 67 The effects of protease inhibitors on digestive proteolytic activity in the midgut of raspberry weevil Aegorhinus superciliosus (Coleoptera: Curculionidae).

Abstract 69

4.1. Introduction 69

4.2. Materials and methods 71

4.2.1. Insects 71

4.2.2. Sample preparation 71

4.2.3. Effect of pH on total proteolytic activity 72

4.2.4. Effect of temperature on total proteolytic activity 72

4.2.5. Specific proteolytic activity 72

4.2.6. Enzyme kinetics 73

4.2.7. Inhibition assays 73

4.2.7.1. In vitro assays 74

4.2.7.2. In vivo assays 74

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4.3. Results 75

4.4. Discussion 78

4.5. Acknowledgement s 81

CHAPTER 5 82

General Discussion and Conclusions Remark

5.1. General Discussion 83

5.2. Conclusions Remark 98

5.3. References 102

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CHAPTER I

General Introduction

1

1.1 General Introduction

Despite advances in strategies for the control of pests and plant diseases, global food supply continues to be threatened by the constant increase of pathogens and pests (Garelik, 2002;

Waage and Mumford, 2008; Rajendran and Singh, 2016; Easwar Rao et al., 2017; Zaynab et al.,

2018). Insect pests are one of the most important biotic stresses for agriculture. They are responsible for a severe reduction in crop yield due to the extensive use of chemical pesticides, affecting beneficial insects and pollute water and environment (Sharma et al., 2000; Ferry et al.,

2004; Bhupendra Kumar, 2016; Velasques et al., 2017; Sharma et al., 2018). Based on their food habits, insect pests are categorized into two types: specialists and generalists or polyphagous insects (Broadway, 1996; Lampert, 2012; Ali and Agrawal, 2012; Schweizer et al., 2017).

Pesticides protect effectively, but their applicability may be affected by adverse effects on the environment and the emergence of insects and pathogen resistance (Bhupendra Kumar, 2016).

Therefore, much effort has been invested toward understanding the mechanisms of innate resistance in plants, since they can activate an array of very effective inducible defense responses such as genetically programmed suicide of the infected cells (hypersensitive response

HR)(Hilker and Fatouros, 2016), tissue reinforcement and antibiotic production in the site of infection (Bowles, 1990; Hammond-Kosack, et al., 1996; Pieterse et al., 2014; Jaber and Ownley,

2018). These local responses may, in turn, trigger a long-lasting systemic response (systemic acquired resistance, SAR) preparing the plant for resistance against a broad spectrum of pathogens

(Dong, 2001; Metraux, 2001; Ökmen and Doehlemann, 2014; Wenig et al.,2019).

2

Plant have resistance factors for direct defense against herbivorous insects that comprise, plant traits that negatively affect insect preference (Jones and Hammond-Kosack, 1997; Howe and

Schaller, 2008; Zaynab et al., 2018; Kirsch et al., 2020). ), there is a large number of these strategies, which are used by them when being attacked by pathogens (Baldwin, 2001; Stotz et al., 2013; Zaynab et al., 2018), insects, herbivores or when subjected to adverse conditions

(Kogan, 1986; Howe and Schaller, 2008, Fürstenberg-Hägg et al., 2013; Zaynab et al., 2018 ). The characteristics of the plants that are important for resistance to attack by herbivores are complex and operate in many spatial scales involving direct and indirect defenses (Karban and Baldwin,

1997; Baldwin, 2001; Giri et al., 2005; Wu and Baldwin, 2009). Unlike animals, plants lack a defense system based on antibodies, so their protection is based on physical characteristics and on chemical compounds they synthesize (Blanco-Labra and Aguirre, 2002; Frérot et al., 2017), among them, the production of secondary metabolites (SM) (Sepulveda et al., 2003; Lay and

Anderson, 2005; Olivera et al., 2007; Ökmen and Doehlemann, 2014; Purrington, 2017; Rosado-

Aguilar et al., 2017; Kirsch et al., 2020).

The SM are structurally diverse and many are distributed among a limited number of plant species, some of them seem to have a key role in the protection of plants as attractants for pollinators and seed-dispersing animals, as allelopathic agents, protective UV radiation and signal molecules on the formation of root nodules of nitrogen-fixing leguminous plants. They are low molecular weight compounds that not only have great ecological importance because they participate in the adaptation processes of plants to their environment, but also because the active synthesis of secondary metabolites is induced when plants are exposed to adverse conditions such as: a) consumption by herbivores (arthropods and vertebrate), b) attack by microorganisms

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(viruses, bacteria and fungi), c) competition of land space, light and nutrients between different plant species and, d) exposure to sunlight or other abiotic stresses (Croteau et al., 2000; Zaynab et al., 2018). Secondary metabolites are also of interest, because of their use as colorants, fibers, gums, oils, waxes, flavorings, drugs, perfumes, pharmaceuticals and as potential sources of new natural drugs, antibiotics, insecticides and herbicides (Croteau et al., 2000; Dewick, 2002;

Jamwal et al., 2017; Pramanik et al., 2019).

Secondary metabolites such as alkaloids, terpenoids and phenolic substances can be found constitutively in tissues, although others only appear as the primary stimulus response; therefore, they are known as induced substances, examples of them are phytoalexins and defense proteins

(Bowles, 1990; Chrispeels and Sadava, 2003; Zaynab et al., 2018; Ferreira et al., 2019; Abdul et al., 2020). These compounds of protein origin constitute a major source of defense against insect pests and microorganisms, not only for their high specificity and efficiency, but also because some of them are highly regulated, and its synthesis responding to attack by predators or pathogens. Within these induced substances protease inhibitors are included, (Bowles, 1990;

Jongsma and Bolter, 1997; Bateman and James, 2011; Kuwar et al., 2015; Grossi-de-Sá et al.,

2017; Ferreira et al., 2019; Singh et al., 2020), proteins that are present in several tissues of animals, plants and microorganisms (Laskowski and Kato, 1980; Ryan, 1990; Biggs and McGregor, 1996;

Lawrence and Koundal, 2002; Bateman and James, 2011; Zhao and Yaw Ee, 2018; Marathe et al., 2019; Pramanik et al., 2019; Hellinger and Gruber, 2019). Likewise, the protease inhibitors are small proteins of plant origin, that have been described mainly in storage tissues such as seeds or tubers, and have also been identified in the aerial parts of the plant (De Leo et al., 2002;

Fernández et al., 2006; Bateman and James, 2011; Jamal et al., 2013; Zhu-Salzman and Zeng,

2015; Mohanraj et al., 2018; Zhao and Yaw Ee, 2018; Marathe et al., 2019; Melo et al., 2019),

4 that can be induced in response to damage caused by arthropods and pathogens (Ryan, 1990;

Farmer and Ryan, 1992; Ryan, 2000; Orozco-Cardenas et al., 2001; Kant et al., 2015; Rustgi et al.,

2017; Saddique et al., 2018; Singh et al., 2020; Kirsch et al., 2020) (Fig.1.1).

Fig. 1.1 Mode of action of plant defense proteins in the herbivore gut. Plants produce special proteins that are co-ingested and interfere with digestive processes in the gut. Proteinase inhibitors and peptidases inhibit the arthropod’s proteases. Digestive processes of the herbivore are shown in red and counter-measures of the plant in green (modified from Kant et al., 2015).

These inhibitors have been extensively studied to elucidate their structural and functional properties (Bode and Huber, 1992; Rasoolizadeh et al., 2016; Grossi-de-Sa et al., 2017). These inhibitors are classified into families and subfamilies based on amino acid sequence, structure and specificity (Blanco-Labra and Aguirre, 2002; Rustgi et al., 2017; Mohanraj et al., 2018;

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Marathe et al., 2019; Hellinger and Gruber, 2019), which have also been classified according to the type of enzyme that inhibits or according to the mechanism of catalysis and the amino acid present in the active site (Ashouri et al., 2017; Rustgi et al., 2017; Clemente et al., 2019; Singh et al., 2020):

Serine proteases inhibitors: T h e most protease inhibitors known and characterized so far and at the same time the most studied belong to this group (Laskowski and Kato, 1980; Bode and

Huber, 1992; Bode and Huber, 2000; Blanco-Labra and Aguirre, 2002; Van der Hoorn and

Jones, 2004; Wan et al., 2014; Xu et al., 2018; Clemente et al., 2019; Gurumallesh et al., 2019 ).

They include relatively small proteins, c o n t ai nin g between 29 and around 190 amino acid residues (Laskowski and Kato, 1980; Bode and Huber, 1992; Rustgi et al., 2017; Marathe et al.,

2019) and include inhibitors known as Bowman-Birk and Kunitz, which constitute the main components in seeds and legumes such as soybeans (Xu et al., 2018; Clemente et al., 2019).

All of them have a characteristic conformation, but they are also independent in structure.

Cysteine protease inhibitors: These inhibitor enzymes have been identified in vertebrates, invertebrates and plants (Oliveira et al., 2003; Marathe et al., 2019), and have been analyzed in greater detail in rice (Blanco-Labra and Aguirre, 2002; Marathe et al., 2019). They have a molecular mass between 5-87 kDa. and they do not contain free cysteine residues. Based on their structural similarities and their amino acid sequence, they are classified into four families: cystatins, stefin, kininogen and plant cystatins or phytocistatines (Rustgi et al., 2017; Melo et al.,

2019; Gurumallesh et al., 2019).

Aspartic protease inhibitors: They are small peptide consisting of 38-39 amino acid residues,

6 have a molecular mass in the range of 20–22 kDa and contain up to two disulfide (S–S) bonds

(Marathe et al., 2019). Aspartic proteases have relatively few inhibitors. The complex of the diazoacetil-norleucina methyl ester with copper ions inactivates most of them, but "pepstatin", of microbial origin, is the most effective (Umezawa, 1976; Van der Hoorn and Jones, 2004; Marathe et al., 2019).

Metallo protease inhibitors: Are a class of proteolytic enzymes that contain a catalytic metal ion at their active site, which aids in the hydrolysis of peptide bonds leading to protein and peptide degradation (Rustgi et al., 2017; Agíc et al., 2018). They are not of plant origin.

The first evidence of the possible insecticide role of the protease inhibitors from plants was observed when larvae of certain insects were unable to develop normally in products derived from soybean (Lipke et al., 1954). Years later, Green and Ryan (1972) demonstrated that potato

(Solanum tuberosum) and tomato (Lycopersicum esculentum) leaves were able to accumulate protease inhibitors against the damage caused by the attack of insects, such as a response to mechanical damage. In a similar study, Gatehouse et al., 1979 suggested that the resistance in a cowpea crop (Vigna unguiculata) to the attack of the weevil Callosobruchus maculatus was due to the high concentration of inhibitors in this crop.

The use of these inhibitors, whose objective is to affect the digestive enzymes of insects, has been evaluated in several studies when it refers to control pest insects (Mendoza-Blanco and Casaretto,

2012; Zhu-Salzman and Zeng, 2015; Rasoolizadeh et al., 2016; Grossi-de-Sá et al., 2017; Mohanraj et al., 2018; Marathe et al., 2019; Clemente et al., 2019; Kirsch et al., 2020). Its potential has already been demonstrated by assays of incorporation in the diet or in the studies of in vitro

7 inhibition of digestive proteases. In recent years, DNA sequences that encode different protease inhibitors have been incorporated into the genome of different and important plants as cereals, rapeseed, tobacco and potato, and the effects of protection have been obtained in some cases, mainly against fitonematods and Lepidoptera pests (Leple et al., 1995; Urwin et al., 1995;

Koiwa et al., 1997; Urwin et al., 1998; Schuler et al., 1998; Lecardonnel et al., 1999; Ussuf et al., 2001; Oliveira et al., 2007; Abd El-latif, 2015; Kuwar et al., 2015; Zhu-Salzman and Zeng,

2015; Grossi-de-Sá et al., 2017).

Several tests have shown that these proteins may retard growth and development of the insect as well as cause starvation and death of the same it (Hilder et al., 1987; Koiwa et al., 1998; Schuler et al., 1998; Hilder and Boulter, 1999; Mosolov et al., 2001; Grossi-de-Sá et al., 2017; Kirsch et al., 2020; Abdul et al., 2020).

These inhibitors have different modes of inhibition, for instance blocking directly or indirectly the active site of proteases (Habib and Fazili, 2007; Marathe et al., 2019; Clemente et al., 2019). It is believed that the mechanism of action of these inhibitor proteins of proteases on insects begins in the intestine with the activation of a feedback system that causes hypersecretion of digestive enzymes and consequent appetite decrease producing a loss of essential amino acids that causes finally a protein deficiency (Broadway et al., 1986; Abd El-latif, 2015; Ashouri et al., 2017;

Hellinger and Gruber, 2019). According to Van der Hoorn and Jones (2004), protease inhibitors have been expressed in the cytosol or administered extracellularly. Similarly, Kant et al., 2015 explain that when plants recognize herbivores, chemical signals are transmitted through tissues and jasmonate stimulates the increase in the accumulation of secondary metabolites (phytotoxins) among these protease inhibitors. The location, where supposedly these protease inhibitors act

8 in the plant cell, is summarized in Fig. 1.2.

Figure 1.2. Site location inhibitors and proteases in a plant cell. PIs are expressed in the cytosol or extracellularly (Kant et al., 2015).

For feeding, insects need enzymes that allow them acquire the essential amino acids for their growth and development, and, like other organisms, they use proteases to digest proteins that are ingested with food (Xu and Qin, 1994; Dunse et al., 2010a; Dunse et al., 2010b; Kuwar et al.,

2015; Clemente et al., 2019). Digestive proteases are enzymes that catalyze the breakdown of peptide bonds and are found in abundance in the midgut of the insect (Jongsma and Bolter, 1997;

Preciado-Rodríguez et al., 2000). For a long time, it was thought that insects and vertebrates contained only serine protease type trypsin and chymotrypsin and aspartic proteases type pepsin

(Mc Farlane, 1985).

The type, which a specific protease belongs to, is determined by the pH range of activity, by

9 its similarity with proteases completely characterized and by its sensitivity or susceptibility to different inhibitors (North, 1982). Different types of proteases have been isolated from the digestive system of insects (Applebaum, 1985; Blanco et al., 1996) (Table 1.1). However, some proteases differ considerably in their most important characteristics, such as their location, optimal pH of activity, and kinetic and thermal stability, among others (Blanco et al., 1996, Gurumallesh et al., 2019).

Table 1.1. Summary of some characteristics of protease groups.

Protease Organism pH Molecular References Weight (kDa)

Serine Eukaryotes, 6-11 18-35 Antão and Malcata, 2005; Viruses, Bacterias Terra and Ferreira, 2012; Gurumallesh et al., 2019.

Cisteine Procariontes and 4-7 20-37 Turk and Bode 1991; Eukaryotes Gurumallesh et al., 2019

Aspartic Insects and 2-6 30-48 Sielecki et al.,1991; mushrooms Gurumallesh et al., 2019

Metallo Eukaryotes, 5-9 19-74 Caffini et al., 1988; Santos Bacteria and 2007; Gurumallesh et al., Mushrooms 2019

According to various authors (Jongsma et al., 1996; Zhu-Salzman and Zeng, 2015; Terra and

Ferreira, 2020), a wide range of insects of different orders (especially Lepidoptera and

Coleoptera), uses different classes of proteases (serine, cysteine and aspartic proteases) to digest proteins. In studies carried out in larvae of diverse species, Terra and Ferreira (1994; 2020), and Terra et al., (2019), found that serine proteases such as trypsin and chymotrypsin are the

10 dominant ones in the digestive system of these insects.

Similarly, Muharsini et al., (2000) reported the existence of serine digestive proteases in

Chrysomya bezziana (Villeneuve). On the other hand, some proteases secreted by the common cattle grub Hypoderma lineatum (Villers), were also characterized and classified as serine proteases. These proteases play a key role allowing the larvae of H. lineatum nurture and migrate through the tissues of the host (Tabouret et al., 2003). In s ome insects including many of the Coleoptera Order (Murdock et al., 1988; Terra and Ferreira, 2020) using cysteine proteases, whereby midguts tend to have a pH in the slightly acidic range, between 5 and 7, whereas the aspartic proteases often found in combination with other proteases are active in the acidic pH range, typically less than 4.5 (Jongsma and Bolter, 1997).

Currently, one of the most important aspects of pest control, is the selective inhibition of the digestive enzymes of insects, since the digestive tract is the region that connects to the insect with its outer side (Bigham and Hosseininaveh, 2010; Clemente et al., 2019), which leads us to consider:

1) the great economic importance to have crops for human beings, 2) that digestion is a phase of the physiology of insects and 3) the fact that most control measures are related to the action of insecticides on the digestive juices found in the intestinal tract of the insects. From that point of view, the control of pests using secondary metabolites as protease inhibitors, which directly affect the diet and therefore the development of the insect is becoming increasingly necessary. Our country is not exempt from the attack of pests on crops, many of which are controlled with pesticides, although this control is not effective.

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An important pest in berries, is Aegorhinus superciliosus, Guérin-Méneville, 1830 (Coleoptera:

Curculionidae), the raspberry weevil (Fig. 1.3), is a native insect in the Southern Cone of America

(Chile and Argentina) whose larvae have powerful jaws with which it cuts and grinds roots and rootlets of plants (Kuschel, 1951; Aguilera, 1988; Carrillo, 1993; Elgueta, 1993; Aguilera, 1995,

Cisternas et al., 2000; Aguilera and Rebolledo, 2001; Zavala et al., 2011; Quintana et al., 2011;

Rebolledo et al., 2012; Huala et al., 2018).

Figure 1.3. Aegorhinus superciliosus raspberry weevil feeding shoots of blueberry.

Oviposition is performed by the female adult in the neck of its host or in the soil surface

(Caballero, 1972), in areas surrounding the plant, covering the eggs with a sticky substance, which would provide protection against desiccation and act simultaneously avoiding predation (Perez,

1994; Cisternas et al., 2000). At birth, the larvae move into the soil to the root zone near the main root of healthy plants (Fig. 1.4). When introduced into the root, a gallery is drilled whose entrance is covered with sawdust and droppings. This gallery comes to the neck of the plant where the larva enters the pupal stage to complete its development; this may cause the

12 death of the attacked plant (Kuschel, 1951; Elgueta, 1993; Cisternas et al., 2000; Aguilera and

Rebolledo, 2001; Zavala et al., 2011; Quintana et al., 2011; Rebolledo et al., 2012; Huala et al.,

2018). When emerging adults feed on stems, buds and fruit, they may cause defoliation, in severe attacks, that, in some cases, can induce death of the plant (Carrillo, 1993) (Fig. 1.5).

Figure 1.4. Damage of A. superciliosus larvae at the root of blueberry plants.

Figure 1.5. Damage caused by A. superciliosus adults in the stem of blueberry plants.

Currently, A. superciliosus control consists mainly of the use of chemical p roducts; however, despite achieving some effectiveness in adults, it has been supplemented by other control methods, such as cultural, biological and physical-mechanical control, and, to some extent, natural

13 control (Parra et al., 2009; Mutis et al., 2009; Aguilera and Rebolledo, 2001; Zavala et al., 2011;

Quintana et al., 2011; Rebolledo et al., 2012; Rodriguez-Saona et al., 2019), without having obtained the best results. As this attack is produced in the soil, direct chemical control with insecticides is practically ineffective. Other limitations of this type of control, when directed to the adult, are the long residual effects and long lack period of products used, which are harmful to the bees that pollinate the crop when applied near flowering period (Aguilera, 1995).

Therefore, the search for new alternatives for the control of this insect is necessary due to the ineffectiveness of the products used and the environmental problems caused by chemical insecticides. A new approach could focus on the physiology of this insect, for which it is necessary to morphologically and histologically characterize the digestive system, since it has been proven that the intestine is a very large and relatively unprotected interface between the insect and its environment. The final digestion of all nutrients takes place on the cell surface of the midgut, which is responsible for all steps in food processing: digestion, absorption and feces elimination (Dias et al., 2019; Terra and Ferreira, 2020). Therefore, the understanding of this organ is essential to develop control methods that act directly through it, such as the use of secondary metabolites, aimed at affecting the feeding and development of the insect, as is the case with of proteases inhibitors.

In the same way, the proteases present in the midgut of the adult insect must be identified, to later evaluate the protease inhibitors effects on the tissue of the digestive system, on digestive enzymes, on feeding and development of the insect, when ingesting PIs in your food. This approach will allow generate new strategies to carry out adequate integrated management of this pest and achieve efficient and ecologically sustainable control.

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1.2 Hypothesis

Protease inhibitors damage the digestive system tissue, produce an anti-alimentary effect and negatively affect the development in adults of Aegorhinus superciliosus, Guérin-Méneville

(Coleoptera: Curculionidae).

1.3 General Objectives

To evaluate the effect of protease inhibitors on the digestive system tissue, feeding, development and the proteolytic activity of proteases isolated from A. superciliosus.

1.4 Specific objectives

I. To characterize morphologically and histologically the digestive system in adult of A. superciliosus to describe the structure, composition and characteristics of normal tissue in this insect, and use these results to accomplish objective IV. (Chapter II).

II. To determine the effect of protease inhibitors (PIs) on digestive system tissue, feeding and development of raspberry weevil. (Chapter III).

III. To identify the proteases present in the digestive tract of adult A. superciliosus. (Chapter IV)

IV. To evaluate the effectiveness of protease inhibitors on digestive proteases of adults. (Chapter

IV).

15

CHAPTER II

“Morphological and histological characterization of digestive system of Aegorhinus superciliosus raspberry weevil (Coleoptera: Curculionidae)”

Article published in Revista Colombiana de Entomología (2013) 39 (2): 260-266

16

Medel Vivian1, Molina Benedicto2, Seguel Jorge2, Rebolledo Ramón3, Quiroz Andrés4.

1Doctoral Program in Science of Natural Resources, Scientific and Technological Bioresource

Nucleus (BIOREN_UFRO), Universidad de La Frontera, PO Box 54- D, Temuco, Chile.

2 Basic Sciences Department, Laboratory of Histology, Universidad de La Frontera, PO Box

54- D, Temuco, Chile.

3Agricultural Sciences and Natural Resources Department, Applied Entomology Laboratory.

Universidad de La Frontera, PO Box 54- D, Temuco, Chile.

4Chemical Ecology Laboratory, Chemical Sciences and Natural Resources Department, Universidad

de La Frontera, PO Box 54- D, Temuco, Chile

*Corresponding author: [email protected].

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Resumen

Se realizó el estudio morfológico e histológico del sistema digestivo de Aegorhinus superciliosus

(Coleoptera: Curculionidae), plaga de importancia económica en el sur de Chile. Esta especie presenta un sistema digestivo característico para la mayoría de los insectos, siendo similar en machos y hembras, pero de mayor longitud en las últimas, llegando a medir el doble del tamaño del insecto. El sistema digestivo es un tubo largo de diámetro variable, dividido en tres regiones: intestino anterior (estomodeo), intestino medio (mesenterón) e intestino posterior (proctodeo).

Histológicamente presenta un tejido epitelial similar al descrito para otras especies y externamente un tejido muscular, sin diferencias entre machos y hembras. No obstante, presenta características que difieren en otros curculiónidos y coleópteros tales como: ausencia de buche, ubicación y número de los ciegos gástricos, presencia de membrana peritrófica y ausencia de almohadillas rectales.

Palabras clave: Morfología interna. Plaga del arándano. Chile.

Abstract: A morphological and histological study was performed on the digestive system of

Aegorhinus superciliosus (Coleoptera: Curculionidae), an important economic pest in southern

Chile. This species has a typical digestive system for most insects, being similar in males and females, but longer in the latter, being twice the length of the insect. The digestive system is a long tube of variable diameter, divided into three regions: foregut, midgut and hindgut. From the histological point of view, it presents epithelium tissue features similar to the one described for other species, and externally it has a muscle tissue with no differences between males and females.

Nevertheless, it shows features which differ either in other weevils or in beetles such as: absence of crop, the location and number of gastric caeca, presence of peritrophic membrane, and absence of a rectal pad. Key words: Internal morphology. Blueberry

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2.1 Introducción

El burrito del frambueso Aegorhinus superciliosus (Guérin- Méneville, 1830) (Coleoptera:

Curculionidae) es un insecto nativo del cono sur de América (Chile y Argentina), cuya larva posee poderosas mandíbulas con las cuales corta y tritura raíces y raicillas de sus hospederos, horadando finalmente la raíz principal cerca del cuello donde construye una cámara pupal, actividad larvaria que provoca un detrimento en la planta (Kuschel 1951; Carrillo 1993; Elgueta 1993; Cisternas et al., 2000). Los adultos ocasionan daños a brotes, tallos y frutos, que en ataques graves producen la muerte de la planta (Carrillo 1993).

Pese a la importancia que, desde el punto de vista productivo y económico, representa este insecto existe escasa información referente a su biología, la cual se limita principalmente a los aportes de Reyes (1993), Pérez (1994), Aguilera y Rebolledo (2001) y Parra et al., (2009). En general, las investigaciones realizadas han sido enfocadas al manejo y control de esta especie pero no existen antecedentes sobre la morfología del sistema digestivo de este curculiónido.

Éste, sin embargo, es un área de contacto entre el insecto y el medio ambiente, y el foco de gran parte de los métodos de control de esta plaga (Chapman 1998).

Específicamente la región del mesenterón, presenta un gran interés, ya que las alteraciones en esa zona pueden afectar el crecimiento, desarrollo y todos los eventos fisiológicos que dependen de una adecuada alimentación, absorción y transformación de nutrientes (Mordue y Blackwell,

1993; Mordue y Nisbet 2000). Debido a que no existen antecedentes sobre morfología e histología de los sistemas vitales de este insecto, se planteó como objetivo de este estudio describir morfológica e histológicamente los órganos que comprenden el sistema digestivo,

19 determinando su forma, tamaño y composición celular. Esta investigación aporta información descriptiva básica sobre A. superciliosus que permitirán establecer futuras estrategias de manejo y control.

2.2 Materiales y métodos

2.2.1 Material biológico. Se colectaron manualmente adultos de A. superciliosus entre noviembre 2010 y enero 2011, en una plantación comercial de arándanos orgánicos, ubicada en el Fundo El Roble, sector Martínez de Rozas, Comuna de Freire, Región de La Araucanía, Chile

(38º57’12”S 72º37’54”O).

2.2.2 Lugar del estudio. Los insectos fueron transportados juntos en una caja de plástico transparente al laboratorio de Entomología Aplicada de la Universidad de La Frontera, Temuco,

Chile, donde se alimentaron con hojas de maitén (Maytenus boaria: Celastraceae) y se mantuvieron bajo condiciones controladas de humedad (53% ± 2,3) y temperatura (26 ºC ± 1,1).

2.2.3 Disección. Para el estudio morfológico e histológico, se hicieron disecciones de machos y hembras aletargados en frío (-15 ºC), bajo una lupa estereoscópica binocular marca LEICA MZ75.

La disección se inició con el desprendimiento de las antenas, patas y cortes dorso laterales de tórax y abdomen, fracturando la cutícula hasta llegar a la cabeza y rostrum. Se retiraron los fragmentos de cutícula y de la membrana interna a la cual se encuentra adherido el segundo par de alas, para facilitar la limpieza. Los órganos internos extraídos fueron colocados sobre placas petri con agua destilada (Hosseininaveh et al. 2007; 2009), con pinzas y tijeras de punta fina se realizó la extracción del tejido adiposo circundante para continuar separando el sistema digestivo del resto

20 de los sistemas vitales. El sistema digestivo fue dividido en tres partes (anterior, media y posterior) y luego cada parte se midió bajo una lupa equipada con reglilla micrométrica. Los órganos del tracto digestivo fueron fotografiados con una cámara Nikon para determinar su forma y tamaño, siguiendo la metodología propuesta por Rubio y Acuña (2006).

2.2.4 Preparación histológica de las muestras. Cada parte (intestino anterior, medio y posterior) fue fijada en Bouin acuoso (Baker 1969), durante 48 horas, deshidratada en alcohol y, posteriormente, incluida en paraplast con enfriamiento y solidificación a temperatura ambiente.

Se obtuvieron cortes seriados de 7 µm. de espesor en un micrótomo de rotación Microm HM

335 E para luego ser teñidos con hematoxilina eosina (Uría y Mora 1996).

2.3 Resultados

2.3.1 Descripción morfológica. En adultos de A. superciliosus, el sistema digestivo está constituido por un tubo de diámetro variable y longitud mayor al doble del tamaño del insecto, especialmente en las hembras (Tabla 1). Como en la mayoría de los insectos, este se encuentra dividido en tres regiones bien definidas: intestino anterior o estomodeo, intestino medio o mesenterón e intestino posterior o proctodeo. El intestino anterior ocupa en la hembra el 5%, el intestino medio el 50% y el intestino posterior el 45% de la longitud total. En el caso del macho, los respectivos porcentajes fueron 7, 50 y 43% del largo total del tubo digestivo.

Tabla 2.1. Tamaño (mm) ± desviación estándar (DE) de los diferentes órganos componentes del tracto digestivo del burrito del frambueso Aegorhinus superciliosus.

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2.3.1.1 Intestino anterior. Es un tubo de poca longitud (2,15 mm en la hembra y 2,6 mm en el macho), que se inicia en la boca y termina en el cardias. Comprende la faringe, el esófago y el proventrículo (Fig. 1A). La faringe es un tubo delgado con múltiples tráqueas, que se encuentra entre la cavidad preoral y el esófago (Fig. 1A). El esófago (Figs. 1A, 1C) es un tubo corto, poco diferenciado que no presenta la zona de ensancha- miento conocida como buche y se une al proventrículo (Figs.1B, 1C). Este último es la zona de mayor diámetro y se presenta como una dilatación definida (órgano esférico), siendo la sección más dura y esclerotizada del canal alimentario que culmina en la válvula “cardíaca” o cardias (Fig. 1B), un área de poca longitud que no permite que los alimentos regresen desde el intestino medio al intestino anterior.

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Figura 2.1. Estomodeo. A. Aspecto general del intestino anterior (ia) en el cual se observan: faringe (f) y esófago (e). B. Vista general del proventrículo (pv) y cardias (c). C. Vista general del intestino anterior (ia) donde se observa el esófago (e) y proventrículo (pv) y del intestino medio

(im) donde se visualiza el ventrículo anterior (va).

2.3.1.2 Intestino medio. Posterior al cardias, se encuentra el mesenterón, que comprende dos regiones: ventrículo anterior y posterior. El ventrículo anterior, es una porción que aparece como una diferenciada dilatación en forma de bulbo (Figs. 1C, 2A) (8,88 mm en la hembra y 8,10 mm en el macho), a su vez el ventrículo posterior (Fig. 2B) es una región larga y angosta (11,34 mm en la hembra y 10,5 mm en el macho), que se encuentra externamente tapizada por numerosos ciegos gástricos de tamaño uniforme, que culmina en una constricción denominada válvula pilórica o píloro.

Figura 2.2. Mesenterón. A. Vista general del ventrículo anterior (va). B. Vista general del ventrículo posterior (vp) con restos de alimento.

2.3.1.3 Intestino posterior. Esta porción es un tubo largo (18,30 mm en la hembra y 16,08 mm en el macho), que se inicia con la válvula pilórica de la cual nacen seis tubos de Malpighi (Fig.

23

3A) de gran longitud, que se encuentran rodeando todo el interior del insecto, especialmente el mesenterón y enlazados con un gran número de tráqueas. Los tubos de Malpighi se agrupan en dos: un grupo que contiene cuatro túbulos y el otro con sólo dos túbulos (Tipo II, según la clasificación de Calder 1989).

A continuación, justo después de la válvula pilórica se encuentra el íleon (Figs. 3A, 3B), que es la porción más larga del proctodeo (6,54 mm en la hembra y 6,38 mm en el macho) y presenta un diámetro uniforme en toda su estructura. Posterior al íleon se encuentra el colon (Figs. 3B,

3D), porción más corta (3,36 mm en la hembra y 2,46 mm en el macho) pero voluminosa. El recto

(Figs. 3B-3D), es la última zona y presenta un área de mayor diámetro formando la ampolla rectal

(Figs. 3C, 3D), la cual concluye en una pequeña porción denominada saco anal (Figs. 3C, 3D). Por

último, se encuentra el ano (Fig. 3D), que es la válvula encargada de evacuar los desechos del insecto al exterior y que termina en una zona más estrecha.

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Figura 2.3. Proctodeo. A. Vista de la unión del mesenterón con el proctodeo: ventrículo posterior

(vp), píloro (p), tubos de Malpighi (tm), ileon (I). B. Vista general del proctodeo, donde observamos el ileon (i), colon (c) y recto (r). C. Vista ampliada del recto donde se observa la ampolla rectal (ar) y el saco anal (sa) con desechos. D. Vista lateral del intestino posterior (ip) con todas sus partes, colon (c), ampolla rectal (ar), saco anal (sa) y ano (a).

2.3.2 Descripción histológica

2.3.2.1 Intestino anterior. Está compuesto, desde el exterior hacia el interior, por una capa de músculos circulares, seguida de una capa relativamente gruesa con músculos longitudinales que se continua en una capa de epitelio simple plano, con la presencia de tráqueas y células con núcleos centrales redondeados, con la cromatina en corpúsculos y un citoplasma acidófilo denso y esponjoso (Fig. 4A). Internamente se encuentra un epitelio simple cilíndrico, cuyas células presentan un núcleo ovoide a un mismo nivel en la región basal. Finalmente, en contacto directo con el lumen, se observa la íntima, una capa delgada, densa, amorfa y plegada hacia el interior y el epitelio muscular (Fig. 4B). El proventrículo está formado por músculos circulares que son responsables de la constricción del mismo durante el filtrado de los alimentos.

25

Figura 2.4. Intestino anterior. A. Corte transversal donde se observa los músculos circulares (mc), los músculos longitudinales (ml), las tráqueas (t), el lúmen (l). B. Vista ampliada del intestino anterior, donde observamos los músculos circulares, el epitelio muscular (em), el epitelio simple

(es) y la capa íntima (i) (40x y 100x).

2.3.2.2 Intestino medio (Fig. 5). Sobre el epitelio se ordenan dos capas musculares: primero externamente una de músculos circulares y otra interna de músculos longitudinales. Entre los haces musculares se acomodan las tráqueas y una membrana de tejido conectivo. Las células más características del epitelio en este insecto, son cilíndricas con microvellosidades, formando un borde estriado que delimita el lumen.

2.3.2.2.1 Ventrículo anterior. La pared del mesenterón comprende dos capas de músculos estriados: externa (circular) con fibras continuas e interna (longitudinal), separadas por espacios intersticiales. Luego se continúa en un epitelio simple cilíndrico de células columnares, altas y delgadas y un núcleo ovoide, claramente visible (Fig. 5A), que está constituido por células regenerativas, con citoplasma fuertemente acidófilo y un núcleo con cromatina dispersa, cuya función sería la absorción y secreción, que descansa sobre una membrana basal delgada. En estas células epiteliales se puede observar la presencia de leves invaginaciones con microvellosidades en la zona apical. El intestino medio carece de íntima cuticular y la parte más profunda, aquella que rodea el lumen o cavidad por donde circula el alimento digerido, está formada por una delgada capa de células epiteliales denominada membrana peritrófica, altamente permeable, ya que permite el paso de la secreción gástrica que va a penetrar en la masa de los alimentos. Entre el epitelio y la membrana peritrófica encontramos un espacio constituido por tejido conectivo.

26

2.3.2.2.2 Ventrículo posterior. Las células características del epitelio de revestimiento de esta sección, son cilíndricas con microvellosidades, formando un borde estriado que delimita el lumen

(Fig. 5B). Las microvellosidades también están presentes en el borde interno. El alimento en el interior del lumen está rodeado por la membrana peritrófica.

Figura 2.5. Intestino medio. A. Vista general de las células columnares del epitelio simple del ventrículo anterior (100x). B. Vista general del ventrículo posterior donde se observan los ciegos gástricos (cg), el epitelio del ventrículo (ev) y el lúmen (l) (40x). C. Vista ampliada de la estructura circular de un ciego gástrico formada por células cilíndricas (c) con su respectivo núcleo (100x).

D. Vista de la abertura independiente de un ciego gástrico hacia el lumen del intestino posterior

(100x).

2.3.2.2.2.1 Ciegos gástricos. El epitelio del ventrículo posterior se caracteriza por la presencia de ciegos gástricos, que se abren en forma independiente hacia el lúmen del intestino y se encuentran distribuidos en forma regular a través del ventrículo posterior (Fig. 5C). Muestran una pared con

27 capas musculares similar a las del canal alimentario; sobre la capa muscular presentan una estructura circular formada por células cilíndricas, con núcleos redondos situados centralmente con el polo distal más angosto bordeando el lumen (Fig. 5D).

2.3.2.3 Intestino posterior (Fig. 6). En general el intestino posterior está constituido por una pared con abundantes músculos circulares y en la superficie luminal está revestido por la capa íntima que se originaría en la región pilórica.

2.3.2.3.1 Ileon. Está constituido externamente por una capa cerosa de células epiteliales planas, que protegen la pared intestinal, a continuación, presenta una pared externa de músculos circulares y una capa interna de músculos longitudinales (Fig. 6A), además se observa un epitelio de revestimiento simple cilíndrico cuyo citoplasma provoca protrusiones hacia el lumen, lo cual forma vellosidades intestinales (Fig. 6B).

Figura 2.6. Intestino posterior. A. Corte transversal del ileon donde se aprecian los músculos circulares (mc), músculos longitudinales (ml), el epitelio (e), las protrusiones del citoplasma (pc)

(40x). B. Vista ampliada del íleon con el epitelio (e), los músculos circulares (mc) y los músculos longitudinales (ml) (100x).

28

2.3.2.3.2 Colon. Porción tubular del proctodeo que está revestido externamente por una pared de células musculares y presenta en su parte interna ondulaciones y pliegues invaginados (Fig. 7A) con presencia de vellosidades que posiblemente estén recubiertas por células secretoras de enzimas.

La porción del colon que está próxima al recto no presenta vellosidades. Esto indica que, en general, el proctodeo tiene la misma estructura, con una pared cubierta por músculos, lo cual ayudaría a darle la forma y a realizar la función de esta región. El revestimiento interno de este órgano está formado por tejido epitelial simple cilíndrico (Fig. 7B) ya que las células presentan contornos rectangulares y la altura de estas es mayor que la que presentan las células del epitelio simple cúbico.

Figura 2.7. Intestino posterior. A. Corte transversal del colon donde se aprecian las células musculares (cm), el epitelio cilíndrico simple (ecs), pliegues invaginados (pi) (40x). B. Vista ampliada de un pliegue del íleon con células secretoras (cs), núcleos (n) y la capa íntima (i) (100x).

2.3.2.3.3 Recto. La pared interna del recto aparece muy plegada y externamente rodeada por una capa desarrollada de músculos circulares y longitudinales que dan forma y ayudan al funcionamiento de esta región (Fig. 8A). Los longitudinales están formados por células planas,

29 delgadas, con núcleo central, alargado, cromatina laxa, con uno o más nucléolos y citoplasma uniforme (Fig. 8B), no se aprecian almohadillas rectales que son comunes en otros insectos. Las células de estos músculos tienen capacidad contráctil, lo que permitiría que los desechos sean expulsados al exterior. Internamente entre las fibras musculares y el detritus presenta un recubrimiento, formado por un epitelio cúbico simple con células esféricas y núcleo ovoide heterocromático y citoplasma basófilo (Fig. 8C).

Figura 2.8. Intestino posterior. A. Corte transversal del recto donde se aprecian los músculos circulares (mc), los músculos longitudinales (ml), el epitelio (e), los pliegues del recto (pr) (100x).

B. Vista ampliada de las fibras musculares del recto (fm) con sus células planas (cp), el epitelio (e) con células y núcleo claramente visible (n) (100x). C. Vista ampliada de la fibra muscular (fm) con células planas y núcleos claramente visibles (n) (100x).

30

2.4 Discusión

El sistema digestivo de A. superciliosus está constituido por un tubo, por lo general, recto o en espiral que se extiende desde la boca hasta el ano, con tres secciones principales que tienen diferentes orígenes embrionarios: el intestino anterior o estomodeo, el intestino medio o mesenterón y el intestino posterior o proctodeo, lo que concuerda con lo reportado por Borror y

DeLong (1969), Belkin (1976), Snodgrass (1993), Chapman (1998) y Terra y Ferreira (2003), y similar a lo observado en otros coleópteros tales como: Dendroctonus spp. (Díaz et al., 2000, 2003;

Silva-Olivares et al., 2003), D. parallelocolli Chapuis, 1869, D. hizophagus Thomas y Bright,

1970, D. valens Leconte, 1859, (Díaz et al., 1998), Trypodendron lineatum (Olivier, 1795),

Gnathotichus retusus (Leconte, 1868), G. sulcatus (Leconte, 1868) (Schneider y Rudinsky 1969),

Cosmopolites sordidus (Germar, 1824), Metamasius hemipterus (Linneaus, 1758), M. hebetatus

(Gyllenhal, 1838) (Rubio y Acuña 2006), Ips pini (Say, 1826) (Hall et al., 2002), Scolytus multistriatus (Marsham, 1802) (Baker y Estrin 1974), Compsus sp. (Rubio y Acuña 2007),

Rhynchophorus palmarum (Linnaeus, 1758) (Sánchez et al., 2000), Phyllophaga anxia (Leconte,

1850) (Berberet y Helms 1972), Anthonomus grandi Boheman, 1843 (MacGown y Sikorowski

1981) e Hyperia postica (Gyllenhal, 1813) (Tombes y Marganian 1967). Estas estructuras también han sido observadas en otros insectos tales como: Anticarsia gemmatalis (Hübner, 1818) (Levy et al., 2004), Apis mellifera Linnaeus, 1758 (Aljedani et al., 2010), Tropidacris collaris (Stoll, 1813)

(Wanderley-Teixeira et al., 2006), Nasutitermes takasagoensis (Shiraki, 1911) (Tokuda et al.,

2001).

Con respecto al intestino anterior, Chapman (1998) y Terra y Ferreira (2003) indican que se encuentra subdividido en diversas partes funcionales: faringe, esófago, buche y proventrículo. En

31

A. superciliosus encontramos las mismas partes excepto el buche, lo cual coincide con la descripción de Sánchez et al., (2000) para R. palmarum, pero difiere de lo reportado por Rubio y

Acuña (2006) para C. sordidus, M. hemipterus y M. hebetatus, Rubio y Acuña (2007) para

Compsus sp. y por Rubio et al., (2008) para Hypothenemus hampei (Ferrari, 1867), debido a que estos insectos presentan un buche fácilmente reconocible. Según Terra (1990), esto se debería a que, en la evolución de los coleópteros, se produjo una gran reducción o pérdida del buche.

Además, Toro et al., (2009) explican que la porción terminal del esófago, fuertemente musculosa, llamada proventrículo no está bien diferenciada en especies de alimentación líquida (pulgas), pero en el caso de A. superciliosus, que tiene un alimento sólido, está claramente definida, lo que muestra la relación del desarrollo del proventrículo con el tipo de alimento ingerido por el insecto.

En el mesenterón de A. superciliosus, se distinguen dos regiones bien definidas: ventrículo anterior y ventrículo posterior, particularidad que está presente en otros coleópteros tal como lo señalan

Wigglesworth (1974) y Crowson (1981), quienes indican además que la estructura y función del mesenterón guarda relación con el tipo de alimento ingerido. De acuerdo a lo anterior, Sánchez et al., (2000) indican que el intestino medio de R. palmarum tiene cierta similitud de forma y tamaño con A. grandis, C. sordidus, M. hemipterus y M. hebetatus ya que Macgown y Sikorowski (1981) y Rubio y Acuña (2006), señalan que en estas especies la porción anterior del mesenterón aparece como una diferenciada dilatación en forma de bulbo ovoide y la porción posterior es un tubo delgado, de menor diámetro, lo cual coincide con lo encontrado en A. superciliosus.

El ventrículo anterior presenta un epitelio columnar similar al encontrado en P. anxia (Berberet y

Helms 1972), R. palmarum (Sánchez et al., 2000) y T. collaris (Wanderley-Teixeira et al., 2006).

Además presenta un borde interno cubierto por microvellosidades, descritas por Snodgrass (1993)

32 y Chapman (1998) para insectos en general. Macgown y Sikorowski (1981), reportaron que en A. grandis el ventrículo posterior se encuentra revestido externamente por ciegos gástricos, los cuales contienen bacterias y otros microorganismos del tubo digestivo que producen enzimas y vitaminas.

En este caso A. superciliosus tiene cierta similitud con A. grandis y Dendroctonus frontalis

Zimmerman, 1868 (Díaz et al., 2000), ya que estos autores señalan que varios ciegos gástricos se identificaron en la porción terminal del ventrículo posterior, lo que coincide con nuestros resultados, caso contrario a lo observado en R. palmarum pues según Sánchez et al., (2000) estos serían dos y se encontrarían en el ventrículo anterior. Además Rubio y Acuña (2006) observaron que los ciegos gástricos de C. sordidus, M. hemipterus y M. hebetatus, se encontraban tanto en el ventrículo anterior como posterior lo cual difiere de nuestros resultados y los reportes de otros autores. Internamente los ciegos gástricos presentan una pared con capas musculares similar a las del canal alimentario y poseen una estructura circular, formada por células cilíndricas lo cual concuerda con lo expuesto por Sánchez et al., (2000) para R. palmarum.

Toro et al. (2009) explican que en especies que ingieren tejidos, el epitelio parece estar protegido por una secreción celular que formaría la membrana peritrófica (PM) que reemplazaría de cierto modo la función protectora del mucus secretado en los animales por las células caliciformes. Al respecto Terra et al. (2006) y Bolognesi et al., (2008) explican, que es una estructura anatómica que rodea el bolo alimenticio en coleópteros y en la mayoría de los insectos. En el caso de A. superciliosus, el intestino medio está recubierto por esta membrana, lo cual según Crowson (1981), estaría relacionada con la ingesta de alimentos, ya que sólo los coleópteros que consumen alimentos con alto contenido de sólidos presentarían dicha membrana. Nuestros resultados coinciden con los reportados por Vásquez-Arista et al., (2001) para Prostephanus truncatus (Horn 1878), pero difieren de los reportados por Sánchez et al., (2000) para R. palmarum, Berberet y Helms (1972)

33 para P. anxia, y Chadbourne (1961) para A. grandis ya que en estos insectos no se encuentra presente dicha membrana.

El mesenterón, se encuentra separado del proctodeo por la válvula pilórica o píloro desde la cual nacen seis tubos de Malpighi, largos, uniformes en todo su recorrido y se disponen enlazados sobre el tubo digestivo. Lo anterior coincide con lo informado para R. palmarum (Sánchez et al., 2000),

C. sordidus, M. hemipterus y M. hebetatus (Rubio y Acuña, 2006), Compsus sp. (Rubio y Acuña,

2007), H. hampei (Rubio et al., 2008). Además con lo reportado por Wigglesworth (1974),

Crowson (1981), Calder (1989) y Snodgrass (1993) quienes han registrado entre cuatro y seis tubos de Malpighi en otros coleópteros.

El proctodeo es un tubo largo, pero de menor diámetro que presenta ondulaciones y pliegues invaginados, constituido por una pared rodeada por una capa de músculos circulares y longitudinales que dan forma y participan en el funcionamiento de esta región. Estas observaciones son similares a las descritas por Berberet y Helms (1972) para P. anxia y Crowson (1981) para coleópteros en general. Además está formado por el ileon, un tubo largo y delgado, similar al descrito para Dendroctonus adjuntus Blandford, 1897 (Zuñiga et al., 1994), pero diferente al encontrado en otros coleópteros, donde se describe como corto o bien como un órgano indiferenciado (Chapman 1998). El colon, está revestido externamente por una pared de células musculares y presenta en su parte interna ondulaciones y pliegues invaginados con presencia de vellosidades lo cual concuerda con lo reportado para R. palmarum (Sánchez et al., 2000). La porción del colon que está próxima al recto no presenta vellosidades. Esto indica que en general el proctodeo tiene la misma estructura, con una pared cubierta por músculos, lo cual ayudaría a darle la forma y a realizar la función de esta región. El recto es la última sección del intestino posterior

34 y es un saco amplio y de paredes delgadas (ampolla rectal) que tiene un epitelio cilíndrico que está revestido por células musculares con capacidad contráctil, no presentando almohadillas rectales.

Snodgrass (1993) explica que en algunos coleópteros estos órganos estarían ausentes, difiriendo de lo reportado por Zuñiga et al., (1994) para D. adjuntus. Finalmente, se encuentra el ano, tubo corto adosado al sistema reproductor, lo cual coincide con lo reportado para otros coleópteros

(Wigglesworth 1974; Crowson 1981; Sánchez et al., 2000; Rubio y Acuña 2006).

2.5 Conclusión

Se describe por primera vez la morfología e histología del tubo digestivo del burrito del frambueso

Aegorhinus superciliosus. Los resultados indican que presenta la misma división y estructuras típicas descritas para la mayoría de los insectos y para otros coleópteros de la misma familia. Sin embargo existen diferencias como: ausencia de buche, número y posición de los ciegos gástricos, presencia de membrana peritrófica y ausencia de almohadillas rectales. De acuerdo a los antecedentes presentados y debido a que no existen estudios de la anatomía interna e histología de

A. superciliosus, se considera que esta investigación representa un aporte para el conocimiento de aspectos básicos de este insecto en nuestro país y puede establecer futuras estrategias de manejo y control de esta importante plaga del arándano.

2.6 Agradecimientos

Al Dr. Ramón Rebolledo, al equipo de trabajo del Laboratorio de Histología de la Facultad de

Medicina de la Universidad de La Frontera, al proyecto DI12- 0026 DIUDRO y a CONICYT por la beca de post grado, la cual ha permitido realizar esta maravillosa investigación.

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CHAPTER III

Effects of protease inhibitors on the digestive system, feeding and development of Aegorhinus superciliosus raspberry weevil (Coleoptera: Curculionidae)

36

Effects of protease inhibitors on the digestive system, feeding and development of

Aegorhinus superciliosus raspberry weevil (Coleoptera: Curculionidae)

Medel Vivian1, Rebolledo Ramón2, Quiroz Andrés3

1Doctoral Program in Science of Natural Resources, Scientific and Technological Bioresource Nucleus

(BIOREN_UFRO), Universidad de La Frontera, PO Box 54- D, Temuco, Chile

2Agricultural Sciences and Natural Resources Department, Applied Entomology Laboratory.

Universidad de La Frontera, PO Box 54- D, Temuco, Chile.

3Chemical Engineering Department, Chemical Ecology Laboratory, Universidad de La Frontera,

PO Box 54- D, Temuco, Chile

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Abstract

Blueberry (Vaccinium corymbosum) ranks as one of the most commonly consumed berries for their high antioxidant and beneficial health properties. Therefore, due to the blueberries consumption exports of these berries are growing every year by leaps and bounds, being the fruit with the largest production area of the country. At present, the fruit sector is strongly threatened by the presence of Aegorhinus superciliosus, raspberry weevil (Coleoptera: Curculionidae) considered as the most important pest of this crop. Farmers invest considerable resources it’s control, which consists mainly of the use of chemicals.

In vivo effects of three commercial inhibitors (PMSF, TPCK and SBTI) were evaluated spraying on the leaves of Rubus ulmifolius, for five days before feeding insects, to identify their effectiveness, selectivity and sensitivity about the digestive proteases of the adult. The results indicate that PMSF and TPCK, in respective concentrations tested, exert an antifeedant effect on adult raspberry weevil. Likewise, SBTI inhibitor exerts an insecticidal effect producing

100% mortality of individuals in the highest tested concentration; PMSF causes 10% mortality the same as TPCK. Furthermore, when comparing the histological samples of the digestive tissue of the insect before and after being fed with leaves of Rubus ulmifolius sprinkled with protease inhibitors, it is verified that these inhibitors do not produce any negative effect on the cellular tissue, since the samples taken after the feeding did not present any malformation or destruction of the cells.

According to the results, SBTI was the most effective inhibitor causing 100% mortality of individuals, compared with the other two products applied. On the other hand, mortality was higher

38 in females than in males in the three doses tested. This demonstrates that protease inhibitors may be potential insecticides for the control of Aegorhinus superciliosus.

Keywords: Aegorhinus superciliosus, mortality, feeding deterrent.

39

3.1 Introduction

Due to the good adaptation to climate and soil conditions, growing berries, such as blueberries and raspberries, are prominent crop of the south central region of our country and the production of these berries is mainly for export (Devotto and Gerding, 2001; Abreu et al., 2008; Kong, 2009;

Song and Hancock, 2011). In the case of blueberry (Vaccinium corymbosum) (Fig. 3.1) it is a minor fruit bush, native from North America that belongs to the Ericaceae family and is considered within the group of the berries (Pritts and Hancock, 1992; Aldaba et al., 2016). There are three types: the high bush blueberry, Vaccinium corymbosum, rabbit eye blueberry, Vaccinium ashei and low blueberry, Vaccinium angustifolium (Buzeta, 1997; Plunkett et al., 2018). The United

States is the largest producer and consumer of blueberries, but the demand for this fruit has grown considerably in Europe and Asia (Odepa, 2019).

Figure 3.1. Growing Vaccinium corymbosum blueberry fruit produced extensively in south central Chile.

In Chile it was introduced in the early eighties and is positioned as one of the most commonly consumed berries, due to its benefits to human health as: its antioxidant properties, anti-

40 inflammatory, anticarcinogenic and preventing urinary tract infections, among others (Ofek et al.,

1996; Wedge et al., 2001; Wang et al., 2005; Aldaba et al., 2016; Plunkett et al., 2018; Stefănescu et al., 2019). In recent years the production of this fruit in our country has experienced a considerable growth, so it is due to blueberry consumption that exports of these berries are growing every year by leaps and bounds.

The North American market covers about 80% exports of blueberries, whereas Europe follows it with 20% and Asia with 3%. The main destination of fresh blueberries remains the United

States of America, where an average price of $ 5.03 per kilogram of exported Fob was obtained

(without total IVV), a market to which 55% of the volume of this exported fruit was sent the

2018/19 season (Odepa, 2019).

In the 2018/19 season, 71 thousand tons of fresh blueberries have been exported, and in the season prior to January 2018, 61 thousand tons were exported, and the 2017/18 season ended with 109 thousand tons of fresh blueberries exported (Odepa, 2019). Due to this, cultivated area has reached over 14,505.9 hectares planted until 2012, which are distributed between Atacama and

Los Lagos regions, being the fruit with the most extensive production area in the south of the country (Odepa, 2019).

At present, the fruit sector is strongly threatened by the presence of insect pests that cause considerable damage to the crop with economic losses. The most important economically pests in blueberries are characterized by rhizophagous (eat roots) (Devotto and Gerding, 2001), and belong mainly to the Curculionidae family (Kuschel 1951; Elgueta, 1993; Cisternas et al.,

2000). Among the different species of this family causing damage to the crop stands,

41

Aegorhinus superciliosus native species considered the most important pest, which has been able to colonize a number of fruit bushes introduced by man as: raspberry, currant, blackberry and zarzaparilla (Elgueta, 1993). Adults cause damage mainly in the aerial part, through the consumption of the cortex in young terminal shoots of the plant and the larvae feed at neck level and roots, causing death of them (Elgueta, 1993, Cisternas et al., 2000; Zavala et al., 2011;

Quintana et al., 2011; Rebolledo et al., 2012; Huala et al., 2018).

Economic importance of this insect was established in 1988, when Aguilera mentions raspberry weevil (RW) as the most important insect pest in plantations of blueberries for the damage it causes. Then, Gonzalez (1989) summarizes its economic importance as occasional in trunks and branches of raspberry or plum root. In turn, Prado (1991) mentions it as an occasional pest in blueberry, raspberry, strawberry, currant, apple, blackberry and sarsaparilla. Meanwhile, Aguilera

(1990) considers this species as potentially quarantine, taking into account it is only found in the

Southern Cone of America (southern Chile and Argentina). Later, Aguilera (1995) cites it again as a pest of economic importance, due to the increase of its population, which is associated with the increase in blueberry plantations in the country. During 1996, the Servicio Agrícola y

Ganadero (SAG) amended the Resolution No. 1910 of 1982, which specified that hatcheries, nurseries and reservoirs must expend pest free plants especially those identified in their payroll.

Among them, Aegorhinus sp. is named as pest in small fruit, specifically berries.

Finally, the European Community (2003) through the Official Journal of the European Union considers the species of A. phaleratus and A. superciliosus as harmful organisms Chile exports to countries that belong to the community.

42

In Chile, farmers invest considerable resources to control this insect, the main control strategy, the use of chemicals. Aguilera (1988; 1992; 1995), explains that non-selective organophosphate insecticides, are the most effective product in controlling adults under semicontrolled conditions; however, Carrillo (1993) recommends the use of insecticidal carbamate family. Nevertheless, these products have a period of lack at least 30 days, being this condition a limiting factor for the management required for blueberry. Another limitation is the long residual effect presented by these insecticides, which are harmful to pollinators when applied near flowering period

(Aguilera, 1995; Aguilera and Rebolledo, 2001; Mutis et al., 2009; Parra et al., 2009; Quintana et al., 2011). Given the drawbacks that insecticides of wide spectrum have, it has been experienced with products that inhibit the formation of chitin, obtaining 95-100% effectiveness depending on the dose. However, these products have no effect on adults (Aguilera, 1995).

The exclusive use of these products does not only result in the rapidly increased resistance to such compounds, but also their non-selectivity affects the balance between pests and natural predators, usually in favour of the pests (Metcalf, 1986; Campos et al., 2019). Due to the deficiencies of traditional synthetic insecticides, agrochemical companies have been progressively changing their management plans, leaving broad-spectrum insecticides and of long persistence that are able to control many pest species simultaneously requiring fewer applications, and preferring products with greater selectivity and shorter period of persistence in the environment (Silva et al., 2002;

Fuentes, 2003). Therefore, botanical insecticides have emerged again as one of the least risky options and more sustainable to phytoprotection after being displaced by inorganic insecticides and later by organic insecticides (Rodríguez et al., 2003; Murray, 2006; Campos et al., 2019).

In addition, we must consider natural control to some extent, which Ripa and Caltagirone

43

(1994) defined as the biological control that occurs without human intervention, being natural enemies important for reducing the harmful organism. This is the case of entomopathogenic fungi and nematodes, which infect larvae, pupae and adults of the genus Aegorhinus causing death

(Gerding, 1999; France et al., 2000). Although the results obtained at laboratory level are satisfactory, at field level they are very dependent on the abiotic conditions as well as to the toxicity of the insecticides and fungicides on these controllers (Molina, 1997; Wu et al., 2014;

Martínez-Hernández et al., 2015).

Similarly, Prado (1991) points to the braconid wasp genus Centistes and the entomopathogenic fungus Metarhizium anisopliae as natural enemies of this insect at the larval stage (Aguilera,

1994); whereas, Carrillo (1993) mentions that this insect has few natural enemies, although the presence of dipteral larvae taquínidae adult’s family is detected. Despite advances in search of

"friendly" alternatives to the environment, specifically semiochemicals responsible for intraspecific and interspecific behaviors (Parra et al., 2009; Mutis et al., 2009; Frérot et al.,

2017; La Forgia and Verheggen, 2018); these records have not been extrapolated to field, so it is necessary to study new methods of controlling this insect. Therefore, with the development of techniques in molecular biology that allow the genetic transformation of plants by recombinant

DNA technology, it has been possible to transform plants with new nutritional properties as well as herbicide tolerant plants resistant to pests and illnesses (Sharma et al., 2000; Cockburn, 2002;

Babu et al., 2003; Khalid et al., 2018; Shah et al., 2018). In recent years, DNA sequences encoding various protease inhibitors have been incorporated into the genome of various plants such as cereals, rape, snuff and potato, and the protective effects were obtained primarily against nematodes and lepidopteran pests (Leple et al., 1995; Urwin et al., 1995; Koiwa et al., 1997;

Urwin et al., 1998; Schuler et al., 1998; Carbonero et al., 1999; Lecardonnel et al., 1999; Ussuf et

44 al., 2001; Bisht et al., 2019).

The physiological significance of the protease inhibitors of plant origin has been questioned for a long time. These inhibitors can be found as original components of plants, particularly in seeds, or can be induced in response to pathogen attack (Birk, 2003; Rodrigues Macedo et al., 2003;

Bateman and James, 2011; Cespedes et al., 2013; Dantzger et al., 2015; Muni Kumar et al., 2017;

Hellinger and Gruber 2019; Saikhedkar et al., 2019).

The presence of a "defense response" of plants to insect attack was first reported by Green and

Ryan (1972). They showed expression and rapid accumulation of the protease inhibitor proteins in potato and tomato leaves in response to a systemic signal appeared as a direct result of injury to the plant leaves, either mechanically or by following insect attack (Broadway and Duffey, 1986a;

1986b). Early insect-resistant transgenic plants were obtained in 1987 and were snuff plants

(Barton et al., 1987; Vaeck et al., 1987) and tomato (Fischhoff et al., 1987), expressing a δ- endotoxin (cry toxin) from the bacterium Bacillus thuringiensis (Berliner), which afforded no protection against lepidopteran larvae. Subsequently, toxin B. thuringiensis was transferred to many plants (Koziel et al., 1993; Schuler et al., 1998; 1999; Hilder and Boulter, 1999; Carbonero et al., 1999; Kumar et al., 2008; Grossi-de-Sá et al., 2015; Rasoolizadeh et al., 2016; Muni Kumar et al., 2017; Khalid et al., 2018). The only insect-resistant transgenic plants that have been commercialized so far are those expressing toxins of B. thuringiensis (Kumar et al., 2008; Sharma et al., 2010; Rasoolizadeh et al., 2016).

In 1987 a gene encoding a trypsin inhibitor was transferred from a legume Vigna unguiculata, a tobacco plant (Hilder et al., 1987). Likewise, they were transferred to other plant genes that confer

45 protection against insects such as α-amylase inhibitors, lectins, proteins chitinase (Lima et al.,

2018) and VIP (Vegetative Insecticidal Proteins) (Carbonero et al., 1993; Ussuf et al., 2001;

Carlini and Grossi-de-Sá, 2002; Babu et al., 2003; Haq et al., 2004; Dively, 2005). Gene expression of protease inhibitors has been detected in the leaves of several species, after they have been affected by any organism, suggesting their role in protecting plants against insect attack and microbial infection (Ryan, 1990; Ussuf et al., 2001; Muni Kumar et al., 2017). These genes have been identified and cloned from a wide range of plant sources including: alfalfa (Mcgurl et al., 1994), tomatoes, potatoes, corn, mustard (Ceci et al., 1995), snuff, rice, sweet potato, soybeans, poplar (Hollick and Gordon, 1995), amaranth (Valdés-Rodriguez et al., 1993), cowpea, and barley.

In addition, these PIs have been described in sunflower, barley, thistles (Cynara cardunculus) and tubers pope (Lawrence and Koudal, 2002). Three major families of plants are known to be rich in these inhibitors: legumes, Solanaceae and Gramineae (Blanco-Labra and Aguirre, 2002;

Yoshizaki et al., 2007; Dantzger et al., 2015).

A large number of these proteins have been described; they only act on predators that attack the plant or grain, producing no effect on others. Therefore, it is important to consider that these alternative mechanisms should not cause damage when humans or animals use it as food (Costa et al., 2019; Saikhedkar et al., 2019). This may be due to their inactivation during the cooking process, or, due to their strict selectivity that allows them recognize as target only the insect or pathogen concerned/involved (Blanco-Labra and Aguirre, 2002).

Interruption of protein digestion in insects by transforming plant genomes with proteinaceous protease inhibitors represents an alternative approach to the control of pests (Reeck et al.,

1997; Parde et al., 2010; Cespedes et al., 2013; Muni Kumar et al., 2017). However, due to

46 variability among insect’s proteases (Wolfson and Murdock, 1990) and to the limited spectrum of activity of these inhibitors (Garcia-Olmedo et al., 1987), expression in plants of a protease inhibitor in particular may not result in a broad- spectrum control.

Therefore, it is necessary to select the appropriate inhibitors for digestive proteases of each particular pest species. This requires knowledge of the proteases in the insect gut and the way they interact with different inhibitors. Furthermore, it has been shown that insects are physiologically adapted to elude protease inhibitors of the plants by enzyme secretion “inhibitor resistant” and by proteolysis to the protease inhibitors of non-target digestive proteases (Broadway, 1996; Jongsma and Bolter, 1997; Muni Kumar et al., 2017).

A program of integrated pest management (IPM) that includes a combination of practical such as: rational use of pesticides, crop rotation, cultural work, the use of transgenic plants with additional protease inhibitors (Jongsma et al., 1996; Michaud, 1997; Costa et al., 2019), and above all, exploitation of inherently resistant plant varieties provides a better option (Meiners and Elden,

1978). The contribution of protease inhibitors as defense mechanisms is based on the inhibition of proteases present in the insect gut; therefore, being anti-metabolic protein, they may interfere with the digestive process of these arthropods (Ussuf et al., 2001; De Leo et al.,2002; Mendoza-Blanco and Casaretto, 2012; Muni Kumar et al., 2017; Hellinger and Gruber, 2019; Saikhedkar et al.,

2019).

Knowledge of IPs and their mechanisms of action represent an interesting alternative to control insects that attack crops. The present study aimed to test the efficacy of three proteases inhibitors, in different doses, included in the diet of A. superciliosus and evaluate their negatives effects on

47 the tissue of the digestive system, feeding and development of the adult insect. The results will be the basis for selecting the appropriate inhibitors to negatively affect this species, thus making an important contribution to sustainable agriculture in our country.

3.2 Materials and methods

3.2.1 Adult insect collection. Adult A. superciliosus were collected manually and randomly in a commercial organic cranberry planting located in El Roble farm, Freire commune (lat.

38º57´12”S; long. 72º37´54”O.) 38 km from Temuco during the period between December

(2012) and January (2013). They were placed in transparent plastic boxes with food, taken to the

Entomology Laboratory at the Universidad de La Frontera and kept at room temperature until dissected.

3.2.2. Plant material. The food substrate for the insects corresponded to Rubus ulmifolius

Schott ( Rosales: Rosaceae) (Fig. 3.2), blackberry leaves approximately 10 cm long, which were extracted from Fundo Maquehue, belonging to the Faculty of Agriculture and Forestry,

Universidad de La Frontera.

48

Figure 3.2. A. superciliosus feeding Rubus ulmifolius leaves.

3.2.3 In vivo assays. 220 insects were kept at room temperature in a clear plastic box prior to the start of the experimental period. Maintenance feeding consisted of Maitenus boaria outbreaks and insects kept fasting for 48 hours before each test application according to Quintana et al., (2011) and Rebolledo et al., (2012) methodology.

For the entire experimental period a 1 maximum and minimum thermometer brand VETO and CIA

®, 1 SUNDO ® hair hygrometer was used, which dealt with for achieving the aim of measuring the environmental conditions of the laboratory.

3.2.3.1 Insect mortality and antifeedant effect determination. Was evaluated according to

Quintana et al., (2011) and Rebolledo et al., (2012) This applies to each experimental unit with 20 repetitions. A total of 220 adult weevils (110 males and 110 females) were placed individually in transparent plastic containers with fresh Rubus ulmifolius leaves A leaf that had previously been

49 treated with inhibitors was placed inside each container, and an adult was added immediately

(one leaflet / pack / day) (Fig. 3.3). Solutions of each of the inhibitors PMSF, TPCK and SBTI with concentrations of 0.1, 1, and 10 mM were prepared in water or ethanol according to solubility. The control consisted of a leaf treated with the corresponding solvent (i.e., water or ethanol). Ten repetitions per sex and per each treatment were used. Every 24 h, the food (a fresh R. ulmifolius leaf) was renewed after the appropriate inhibitor solution had been applied to the new leaf. The number of dead insects was recorded after 24 h in contact with the treated leaf. This procedure was repeated at 48, 72, 96, and 120 h. Once the assay was finished, the average number of dead insects per treatment was determined. In addition, the weight of insects was recorded daily. The data obtained were analyzed with a Fisher test (p < 0.05) using the statistical software Stats Direct v.2.7.8.

To eliminate insect mortality resulting from causes unrelated to the treatment, mortality was corrected using Abbott’s formula (Silva et al., 2002).

Figure 3.3. Assays to evaluate antifeedant effect of protease inhibitors on A. superciliosus.

3.2.4 Dissection. To study the effects of protease inhibitors on the digestive system tissue, the

50 organ of 90 insects used in the mortality and feeding deterrent assays was extracted. Dissections lethargic cold (-15 °C) males and females were made under a LEICA MZ75 stereomicroscope brand. The dissection began with the detachment of antennae, legs and cuts at the back- si de thorax and abdomen, fracturing the cuticle to reach the head and rostrum. Cuticle and the inner membrane, to which the second pair of wings fragments are adhered, were removed to facilitate cleaning. The extracted internal organs were placed on petri dishes with cold distilled water

(Hosseininaveh et al., 2007; 2009), with tweezers and fine tip scissors; extraction surrounding adipose tissue was performed to continue separating the digestive system of other vital systems.

The digestive system was separated into three parts (anterior, middle and posterior) and each part was measured under a magnifying glass micrometer equipped with a micrometric ruler. The digestive tract organs were photographed with a Nikon camera to determine its shape and size, following the methodology proposed by Rubio and Acuña (2006).

3.2.5 Histological preparation of samples. Each digestive system was divided into three parts (foregut, midgut and hindgut), and each of them was fixed in Bouin solution for 48 hours, then the fixative was removed and samples were dehydrated in different graduation alcohol and were included in paraffin with cooling and solidification at room temperat ure. To make the samples serial cuts of 7 µm. were made in a rotation microtome Microm HM 335 E, manually operated, and were placed on a glass slide and then stretched on a platen at 35°- 40°C for 10 minutes. Afterwards, they were placed in a stove for twelve hours in their respective trays. Once dried samples were deparaffinized in xylol for ten minutes, they were moisturized in alcohol

(100% and 96%) for ten minutes and washed with water for 10 minutes to remove any residues.

The cuts were stained with hematoxylin for one minute (nuclear staining), then they were washed

51 for fifteen minutes to eliminate waste and were immersed in a bucket with eosin for two minutes

(cytoplasmic staining) according to methodology Uria and Mora, (1996). Subsequently, they were dehydrated in alcohol (70%, 96% and 100%) for five minutes and they were clarified with xylol for ten minutes, then completing the final assembly with a means of quick inclusion

("entellan") and covering them.

Twenty samples were made per section, which were observed under an electron microscope for histological study. Those samples obtained in the tests carried out in Chapter II of this investigation, considered as control samples, were considered normal. In these samples, a layer of circular and longitudinal muscles formed by thin, flat cells with a central, elongated nucleus, loose chromatin, with one or more nucleoli and uniform cytoplasm, with continuous fibers without cell interruption, were observed. The tissue in general is formed by epithelial cells of the columnar type, with a rounded central nucleus and clearly visible chromatin, without rupture of its nuclear membrane and a dense and spongy acidophilic cytoplasm. In addition, the intima is observed, a thin, dense and inwardly folded layer, formed by a simple cylindrical columnar cell epithelium, with strongly acidophilic cytoplasm and a nucleus with dispersed chromatin. In these epithelial cells, slight invaginations with microvilli in the apical area, forming a striated edge are observed. In contact with the lumen is a thin layer of highly permeable epithelial cells called the peritrophic membrane.

This according to the reports of Zuñiga et al., 1994; Terra and Ferreira, 1994; Nava et al., 2007;

González et al., 2010; Chapman et al., 2013 among others.

3.2.6 Experimental Design. The experimental design was a completely randomized design with

20 replications and five treatments including controls without product (Table 3.1), where the factor of study was the effect of protease inhibitors, and the response variables were mortality and

52 antifeedant effect.

Table 3.1. Treatments in vivo inhibition assays.

TREATMENT CONCENTRATION

Control Water Control Ethanol T1: PMSF 0,1 mM 1 mM

10 mM

T2: TPCK 0,1 mM 1 mM

10 mM

T3: SBTI 0,1 mM 1 mM

10 mM

3.2.7 Data analysis. The statistical analyses were kruskal- wallis to assess antifeedant effect using the SPSS 22.0 statistical software. The percentages of effectiveness for controlling A. superciliosus were calculated for the 24, 48, 72, 96 and 120 hours long of each test.

3.3 Results and discussion

3.3.1 Effect of protease inhibitors on the digestive system. In the present study, the histopathological effects of protease inhibitors on the digestive system of the adult A. superciliosus insect are evaluated. However, it should be noted that studies conducted by other researchers on the effects that PI causes on the tissue of the digestive system of insects fed with these compounds are scarce, and have been carried out in larvae of different stages and not in adults. Most research

53 evaluates the effect of Bacillus thuringiensis toxins on the intestine of pest insect larvae. Despite not coinciding with the state of development of the insects evaluated in the aforementioned studies, these results were compared with those obtained in the present investigation.

The results indicate that the protease inhibitors do not exert any negative effect on the tissue of the digestive system of the adult insect. This due that the histological samples made after feeding the insects with PIs, present morphological characteristics similar to those observed in the histological samples carried out before feeding insects with PIs. Features that are described in chapter II.

The foregut has thick bands of circular muscles (Fig.3.4), similar to the striated muscles of the midgut and a simple cylindrical epithelium of columnar cells with a clearly visible ovoid nucleus, without morphological changes or cell destruction (Fig. 3.5). The epithelial cells are complete, without evidence of lysis or deterioration of their walls, and they have slight invaginations with microvilli in the apical zone and in the peritrophic membrane. In the posterior part of the midgut, the characteristic cylindrical cells with microvilli are observed, forming a continuous striated border that delimits the lumen (Fig. 3.6). These results differ with the statements by Sasaki et al.,

2015, on the effects of the Adenanthera pavonina seed protease inhibitor on the larval digestive system of the Aedes aegypti, which produced morphological changes in the intestine cells, due to the degeneration of the microvilli of the epithelial cells in the posterior region of the midgut, also produced hypertrophy of the gastric caeca cells and an increase in the ectoperitrophic space.

54

Figure 3.4. Proventricle a) Cross section of the proventricle where there are circular muscles (cm), muscular ridges (mr), lumen (l) (10x). b) Extended view of the proventricle with the circular muscles (cm) and muscular ridges (mr) (100x).

Figure 3.5. Anterior ventricle a) Cross section of the anterior ventricle where there are columnar cells (cc), simple cylindrical epithelium (sce), invaginations (i) (10x). b) Extended view of the invaginations (i) with the columnar cells (cc) (40x). c) Extended view of the simple cylindrical epithelium (sce), columnar cells (cc) and core (c) (100x).

55

Figure 3.6. Posterior ventricle a) Cross section of the posterior ventricle where there are gastric caeca (gc), lumen (l) (10x).

In A. superciliosus the gastric caeca are observed, which opens independently towards the lumen of the intestine and is distributed regularly through the posterior ventricle (Fig. 3.7). These present a wall of muscle tissue, surrounded by cylindrical cells, with centrally located round core, with the narrowest distal pole bordering the lumen (Fig. 3.7). In the hindgut a layer of flat epithelial cells is observed, which protect the intestinal wall, an external wall of circular muscles and an internal layer of longitudinal muscles, in addition to a simple cylindrical lining epithelium with intestinal microvilli (Fig. 3.8).

56

Figure 3.7. Gastric caeca a) Extended view of the gastric caeca with cylindrical cells (cc) and core (c) (100x). b) Cross section of the posterior ventricle where there are cylindrical cells

(cc), ventricular ephitelium (ve) (40x).

Figure 3 .8. Hindgut a) Cross section of the ileum where there are circular muscles (cm), longitudinal muscle (lm), the epithelium (e), the cytoplasm protrusions (cp) (10x). b) Extended view of the ileum with the epithelium (e), the circular muscles (cm) a n d longitudinal muscle (lm)

(100x).

Arruda et al., 2003, when testing an ethanol extract of Magonia pubescens in larvae of Aedes aegypti, reported that the main alterations observed were partial or total cell destruction, high cytoplasmic vacuolization and increased ectoperitrophic space. These histopathological alterations, are similar to those observed by Sasaki et al., 2015 in their study, in addition to the same histopathological alterations reported by these authors, were reported by Lahkim -Tsror, et al.,

1983, in a study in which the B. thuringiensis israelensis bacterium, was applied as a primary powder "R-153-78" consisting of crystals, spores and cell debris. These researchers observed damage to the midgut and gastric caeca in treated dead larvae, which presented the disorganized

57 epithelial layer, most of the missing cells and the destroyed peritrophic membrane.

Similar results were obtained by Kabir et al., 2013 in which the effects produced by different doses of Seseli diffusum seed extract on Aedes aegypti were evaluated, and they observed the digestive tract completely altered, with notable cellular hypertrophy, swollen cells with detachment and vacuolated cytoplasm, the apical zone of these cells was empty. In addition, the limits of the peritrophic membrane were damaged, the edge of the tissue was thinner and interrupted and did not cover the entire surface of the lumen of the midgut. The posterior area was completely damaged, although most of the cells had disappeared, some groups of hypertrophic cells were observed. Instead. The control larvae, on the other hand, showed the digestive tract and its limits intact.

Likewise, Ruiz et al., 2004, using the Cry11Bb toxin of Bacillus thuringiensis, on the midgut of

Aedes aegypti, Anopheles albimanus and Culex quinquefasciatus (Diptera: Culicidae) mosquitoes indicated that general histopathological changes included cytoplasm vacuolization, epithelial cell and nucleus hypertrophy, membrane deterioration and cell disintegration, which is consistent with

Arruda et al., 2003 exposures. Other studies carried out by Denolf et al., 1993, when evaluating the effects of two Bt toxins, indicated that 15 minutes after the intoxication of Ostrinia nubilalis larvae with CryIA (b), CryIA (c) and CryIB, epithelial cells exhibited hypertrophy. After 1 hour, cytoplasm vacuolation had occurred and the microvilli of the membrane edge had been destroyed.

Dos Santos Junior et al., 2020, conducted a study aimed at evaluating toxicity, histopathology and cytotoxicity in the midgut cells of the adult Podisus nigrispinus exposed to spinosad, a naturally origin insecticide, produced by fermentation of the Saccharopolyspora spinosa bacterium. The

58 results showed cell degeneration, cytoplasm vacuolization, chromatin condensation and cell release. Cell death in the midgut of this predator occurred by apoptosis, after exposure to the insecticide. It was therefore concluded that Spinosad is toxic to P. nigrispinus and causes histological and cytological damage followed by cell death in the midgut.

The results obtained in studies carried out by various researchers, to evaluate the histopathological effect of some compounds on the digestive system of plague and predatory insects, agree among them, since they cause damage to the internal tissue, either by cell lysis, malformation, cellular hypertrophy, alteration of membranes etc., but differ from those obtained in the present study, because in the tissue of A. superciliosus after being fed with the PIs, no histopathological damage was observed, presenting the foregut, midgut and hindgut completely normal morphology, characteristics that are appreciate in the images presented and that are explained in chapter II.

3.3.2 In vivo effects of protease inhibitors. Several authors have demonstrated that ingestion of proteinase inhibitors in natural or artificial diets can retard growth and development of several species of insect pests, suggesting that these inhibitors may serve as an effective means of controlling insects (Jongsma and Bolter, 1997; Bode and Huber, 2000; Macedo et al., 2003; Macedo et al., 2004; Haq et al., 2004; Abdeen et al., 2005; Park et al., 2005; Habib and Fazili, 2007;

Bhattacharyya et al., 2007; Oliveira et al., 2007; Macedo et al., 2010; Zhao and Ee, 2018; Ferreira et al.,

2018; Mohanraj et al., 2018; Ferreira et al., 2019; Clemente et al., 2019; Marathe et al., 2019; Zhao et al., 2019). However, as mortality is very low, protection is not complete, although high levels of inhibitors were used. There are some studies which show high levels of mortality in some curculionides species, which were obtained with synthetic inhibitors of low molecular weight and microbial origin inhibitors.

59

3.3.2.1 Antifeedant effect of protease inhibitors. The results show that all insects consumed the leaves sprayed previously with inhibitors; therefore, according to the kruskal- wallis test there are no statistically significant differences between control (water) and SBTI treatment (P ≥ 0.05) in the average consumption area Rubus ulmifolius leaves from A. supercilious. Moreover, there were significant differences between PMSF and TPCK treatments (p ≤ 0, 05) with control, being that leaf area consumed by the insects fed with PMSF and TPCK, was less than area consumed by insects in the control (ethanol), which indicates that these inhibitors would have an antifeedant effect on adult raspberry weevil. These results indicate that SBTI no present antifeedant effect on adult of raspberry weevil. However, PMSF and TPCK inhibitors if exhibit this effect, causing the decrease consumption leaf area in the adult.

These results differ from those obtained in other studies since soybean PIs have been tested as an antinutritional factor on insect metabolism and development by several worldwide groups, including resistance against the European corn borer Ostrinia nubilalis (Larocque and Houseman,

1990), the beet armyworm Spodoptera litura (McManus and Burgess, 1995), the tobacco budworm

Heliothis virescens (Johnston et al., 1995) and diamondback moth Plutella xylostella L. (Zhao et al., 2019). However, it has been shown that Spodoptera frugiperda larvae could circumvent the deleterious effects of soybean PIs by increasing the activity of their digestive enzymes or by the synthesis of less sensitive enzymes (Paulillo et al., 2000). Similarly, Lipke et al., in 1954, conducted an in vivo study to evaluate the effect of SBTI on the proteolytic enzymes of

Tribolium confusum, and the results obtained indicated that there was no inhibition of feeding on this insect.

Thus, generalist herbivores, such as S. frugiperda, appear to have developed a mechanism based

60 on the alteration of the set of endopeptidases enzymes to adapt to different host plants, whereas specialist herbivores do not present a wide spectrum of appropriate enzymes to overcome the non- host plants proteinase inhibitor (Broadway, 1997).

Several workers have been able to show directly that proteinase inhibitors can inhibit the activity of insect digestive proteinases in vitro. For example, the soybean trypsin inhibitor (SBTI) has been demonstrated to be effective against the midgut proteinases of the meal beetle, Tenebrio molitor (Applebaum et al., 1964), the trypsin-like enzyme of the tobacco hornworm, Manduca sexta (Miller et al., 1974; Shukle and Murdock, 1983), protease of the black field cricket’

Teleogryllus commodus (Burgess et al., 1994; Sutherland et al., 2002) and an inhibitory effect on all the proteases of diamondback moth Plutella xylostella (Zhao et al., 2019). These observations have been extended to demonstrate that these proteins can retard insect larval development when incorporated into artificial diets. So far, serine proteinase inhibitors have proven to be effective against some Lepidoptera including Ostrinia nubilalis (Steffens et al., 1978; Larocque and Houseman, 1990), Manduca sexta (Shukle and Murdock, 1983), Heliothus zea and Spodoptera exigua (Broadway and Duffey, 1986a), Heliothis armigera and H. assulta (Xu and Qin, 1994),

Heliocoverpa armigera (Johnston et al., 1995; Muni Kumar et al., 2017), Plutella xylostella (Zhao et al., 2019) and coleopteran Callosobruchus maculatus (Gatehouse and Boulter, 1983).

3.3.2.2 Insecticidal effect of protease inhibitors. The results show that there is a relationship between increasing concentrations and the number of specimens killed by the effect exerted by the inhibitors PMSF and TPCK, however, considering the criterion proposed by Silva et al.,

(2002) who indicate as promising only those treatments with a mortality exceeding 40%, this inhibitors not be effective to cause insect death.

61

It can be indicated that the inhibitor SBTI at all tested concentrations was the only effective treatment, as it recorded higher mortality percentage and efficiency from 96 hours after the third application. Assessing the cumulative mortality at 120 hours, the treatment T3 (SBTI) reached

100% mortality, whereas inhibitors PMSF and TPCK showed little effectiveness in all tested concentrations, producing only 10% death of individuals (Table 3.2). Thus, the different applied treatments, have statistically significant differences with control; on the contrary, if we compare mortality between males and females in SBTI, death occurs mainly in females, however in PMSF and TPCK, it occurs mainly in males, these results cannot be compared with other studies, since the effects of these inhibitors separating insects by sex are not evaluated.

Table 3.2. Percentage of mortality of A. superciliosus obtained by protease inhibitors

Mortality (%) Doses Gender

mM

0,1 1 10

PMSF Males 0 0 0

Females 0 0 10

TPCK Males 0 10 10

Females 0 0 0

SBTI Males 80* 90* 50* Females 100* 80* 100*

*Mortality statistically different from control according to Fisher Test (p<0, 05)

62

Our results on adult growth and development are similar to those obtained in studies by McManus and Burgess (1995) and Gatehouse et al., (1997) for SBTI against S. litura and Lacanobia oleracea, respectively.

From the data obtained in this study, a relationship between the effect of the protease inhibitor

SBTI and survival and development of A. superciliosus can be established. SBTI incorporated in the diet caused a high percentage of mortality, indicating that trypsin-like proteases are vital for the growth and survival of the raspberry weevil A. superciliosus. SBTI has previously been shown to be toxic to insects from different orders (Dorrah, 2004; Franco et al., 2004; Silva et al., 2006;

Saadati and Bandani, 2011; Zhao et al., 2019).

These results agree substantially with those obtained by Ortego et al., (1998), which establish a definite relationship between the effect of protease inhibitors and the survival and development of Aubeonymus mariaefranciscae in feeding trials. These authors tested several serine protease inhibitors in vivo on larvae and adults of A. mariaefranciscae to establish their potential as resistance factors. The results demonstrated that survival was strongly reduced and development significantly retarded in larvae fed with an artificial diet containing 0.2% (w/w) of the protease inhibitors using, including SBTI. The mortality rates obtained with these proteinase inhibitors represent an increment of 35–45% with respect to the mortality of the controls, and are among the highest percentages reported so far on curculionides. However, the results obtained in this study exceed the percentage obtained in the research of Ortego et al., (1998), since insect mortality reached 100% in females fed with SBTI. Similarly, Zhao et al., (2019), indicate that SBTI negatively affects the larvae and adults of Plutella xylostella, delaying the growth and development of these states, but does not cause the death of the insect. On the other 63 hand, Purcell et al., (1992) did not find any mortality or stunting associated with high feeding levels of SBBI and SBTI in the cotton weevil. However, Graham et al., (2002) observed that the expression of cowpea trypsin inhibitor in strawberry is effective in protecting roots against larval feeding vine weevil.

Birk and Applebaum (1960) reported in vivo study effects of protease inhibitors of soybean partially purified on red flour beetle, indicating that protease inhibitors inhibited growth of T. castaneum larval. However, further purification resulted in the separation of trypsin and chymotrypsin inhibitors. T. castaneum larvae were fed with increasing amounts of E-64 and SBTI as well as various combinations of the two inhibitors. Mortality ranged from 0 to 4.1% for all treatments except for the combinat ion of E-64 and SBTI, in which mortality was 10%, not statistically different from the control using the Fisher's test (p ≤ 0, 05).

3.4 Conclusion

Protease inhibitors are a large and important group of molecules that are ubiquitously distributed in nature. They play a crucial role in many biological processes by regulating the proteolytic functions of their target enzymes in living organisms. There are several studies that show that PIs are found in food plants, with particular abundance in legumes, but also in cereals and tubers, where they are part of the plant’s defense against pests attack. In current studies several researchers have observed growth retardation, physiological stress and mortality in insects fed with high doses of

PIs, mainly in the larval state.

In this study, the effects of protease inhibitors on digestive tissue, on the growth and development

64 of A. superciliosus, were evaluated for the first time. According to the results obtained, the inhibitors used in the "in vivo" tests PMSF, TPCK and SBTI did not cause negative effects on the tissue of the digestive system of the adult insect, presenting normal morphology and histology, according to the comparison of the samples obtained after feeding the insects with PIs, with the samples obtained in the tests carried out before feeding the insects with these compounds, and which are described in Chapter II. This may be because the histology in adults is different from that found in larvae, and they may be more exposed to damage caused by toxic compounds.

Therefore, in this case, the PIs are not capable of causing destruction of the peritrophic membrane, destruction of the cytoplasm, destruction of vacuoles, or decrease in the size of the microvilli, among other damages.

When evaluating the effects of PIs on the feeding and development of this insect, the results indicated that the inhibitors PMSF and TPCK can be considered anti-alimentary compounds, since the insects fed with these compounds stopped feeding, due to the inhibition of digestive proteases in the midgut, leading to inadequate digestion, poor absorption of essential amino acids, and growth arrest. Despite this, none of these inhibitors managed to cause the death of the specimens.

On the other hand, the SBTI inhibitor did not produce any anti-alimentary effect, since both males and females were fed in the same way as the control individuals. In other studies, in larvae of various species fed artificially with SBTI, this inhibitor significantly delayed the larval period, delayed pupation and the remaining development period compared to the control. This delay was directly proportional with the increase in the SBTI concentration. These studies suggest that the severe retardation in growth and development caused by the inhibitor would provide a much longer period in the development of the insect until the appearance of adults. In this case, the results are

65 even more promising, since the insects fed with SBTI consumed the food in a normal way, finally causing death, both males and females, producing in the latter the highest percentage of mortality.

In general, the inhibitory effects of the different protease inhibitors on feeding and, consequently, on the development of A. superciliosus were varied, causing from an anti-alimentary effect to death, which would indicate that this insect is susceptible to PIs used in the study, especially SBTI.

The knowledge of proteases in the digestive tract of A. superciliosus (chapter IV), helps to understand the biochemical vulnerability of this insect, as well as the possible inhibitors of these proteases. It is therefore necessary to continue evaluating various inhibitors and their effect, in order to contribute to the development of a more efficient and effective control against raspberry weevil.

4.5 Acknowledgements

We thank Dr. Ramón Rebolledo Ranz, from the Laboratory of Applied Entomology and the

National Commission for Scientific and Technological Development (CONICYT) for thesis support through scholarship 24090126.

66

CHAPTER IV

The effect of protease inhibitors on digestive proteolytic activity in the raspberry weevil Aegorhinus superciliosus (Coleoptera: Curculionidae).

Article published in Neotropical Entomology 44: 77-83. DOI: 10.1007/s13744-014-0250-9

67

The effect of protease inhibitors on digestive proteolytic activity in the raspberry

weevil Aegorhinus superciliosus (Coleoptera: Curculionidae).

Vivian Medel1, Rubén Palma2, Darío Mercado3, Ramón Rebolledo4, Andrés Quiroz3, and

3 Ana Mutis

1Doctoral Program in Science of Natural Resources, Scientific and Technological Bioresource

Nucleus (BIOREN_UFRO), Universidad de La Frontera, PO Box 54- D, Temuco, Chile

2 Insect-Plant Interactions Laboratory, Institute of Plant Biology and Biotechnology,

Universidad de Talca, Talca, Chile.

3Chemical Ecology Laboratory, Chemical Sciences and Natural Resources Department

Universidad de La Frontera, PO Box 54- D, Temuco, Chile

4Agricultural Sciences and Natural Resources Department, Applied Entomology Laboratory.

Universidad de La Frontera, PO Box 54- D, Temuco, Chile.

*Corresponding author e-mail: [email protected]

68

Abstract

The raspberry weevil, Aegorhinus superciliosus (Guérin) (Coleoptera: Curculionidae), is an economically important pest of blueberry in southern Chile. The digestive protease activity of adult insects was investigated using general and specific substrates and inhibitors. Enzymatic assays demonstrated the presence of trypsin- and chymotrypsin-like serine proteinases. Furthermore, in vitro assays using phenylmethylsulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) at 0.01 and 0.1 mM showed percentages of enzymatic inhibition between 0 and 16% for PMSF and 67 to

76% for SBTI, whereas in vivo assays indicated that SBTI caused be- tween 50 and 90% mortality in males and between 80 and 100% in females. Our data indicate the presence of serine proteases and suggest that digestive proteases could be a target for the design and development of strategies to control the raspberry weevil.

4.1 Introduction

Aegorhinus superciliosus (Guerin) (Coleoptera: Curculionidae) is native to southern Chile and is also found in the Neuquen district of Argentina (Kuschel 1951). In Chile, A. superciliosus is a pest of blueberries, raspberries, and other berry crops (Aguilera & Rebolledo 2001). Chemical and biological treatments have been used to control this pest. The application of azinphosmethyl or diazinon insecticides to the foliage before and after flowering reduces adult weevil populations. However, sprays are not effective for controlling the root- boring larvae, and long-lasting residual pesticides are not used because of concerns about toxicity to pollinators (Guerrero & Aguilera 1989, Aguilera 1995,

France et al., 2000). In addition, both fungi and entomopathogenic nematodes have been tested as biological control agents of weevil populations (Gerding 1999, France et al., 2000), but the results

69 have been unreliable due to the variability of abiotic conditions and soil (Molina 1997). The manipulation of populations with semiochemicals is another possible method for pest management.

To date, however, only Parra et al., (2009) and Mutis et al., (2010) reported results supporting this possibility, and no field applications have been described.

Due to the problems associated with the continuous application of synthetic insecticides, including environmental and health risks and residues on harvested fruit, alternative strategies of pest management for this weevil are needed. One of the options is based on the selective inhibition of protein metabolism using protease inhibitors.

Protease inhibitors represent one of the most abundant classes of proteins in plants (Macedo and

Freire 2011) and are a component of natural defense mechanisms against herbivores and pathogens

(Stevens et al., 2012). Mickel & Standish (1947) first reported the possible role of plant protease inhibitors as insecticides. These inhibitors have different modes of action, directly or indirectly blocking the active site of the protease (Habib & Fazili 2007). The proposed mechanism by which these proteins inhibit proteases in the insect gut begins with the activation of a feedback system that causes the hypersecretion of digestive enzymes and results in a decrease in appetite. This decrease results in a loss of essential amino acids, ultimately producing a protein deficiency (Broadway &

Duffey 1986a, b, Marinho-Prado et al., 2012). Numerous studies have shown that these inhibitors may retard the growth and development of the insect, as well as causing starvation and death (Hilder et al., 1987, Koiwa et al., 1997, Schuler et al., 1998, Mosolov et al., 2001). Protease inhibitor genes are being used in transgenic crops in combination with other insecticidal genes. The first commercial deployment of a transgenic protease inhibitor was developed in China, where genetically modified cotton varieties expressing Bacillus thuringiensis toxins as an insecticidal protein against lepidopteran

70 larvae were established (Gatehouse 2011).

In insects, as in other animals, proteolytic enzymatic activity can target and destroy essential proteins and tissues to a sufficient extent to cause mortality (Harrison and Bonning 2010). To provide scientific evidence that allows the development of new strategies of control for A. superciliosus based on the use of protease inhibitors, the aims of the current study were (1) to characterize the proteolytic activities of digestive proteases in the midgut of A. superciliosus, (2) to evaluate in vitro the effects of inhibitors on protease activity, and (3) to evaluate the effect of inhibitors on the feeding of adult individuals.

4.2 Material and Methods

4.2.1 Insects

Weevils were collected manually and randomly from a commercial crop of organic blueberries on El

Roble farm, Freire, Araucanía, Chile (38°57′12 S, 72°37′54 W) from December 2012 to January

2013. The weevils were kept in isolation without food for at least 24 h on moist filter paper in petri dishes at 5°C until the performance of the bioassays in the Laboratory of Entomology of

Universidad de La Frontera (Temuco, Araucanía, Chile). This procedure was performed in accordance with Rebolledo et al., (2012).

4.2.2 Sample preparation

Enzyme extract was obtained according to Pereira et al., (2005). Insects were frozen at −18°C, and

71 the midguts were removed and placed on ice. After homogenization in the cold with 0.15 M NaCl

(1:10; w/v), samples were centrifuged at 16,500×g for 10 min at 4°C, and the supernatants were pooled and frozen at −20°C until required.

4.2.3 Effect of pH on total proteolytic activity

Total proteolytic activity and effect of pH were determined according to Pereira et al., (2005), with some modifications. A total of 50 μL of enzyme extract was added to 170 μL of buffer (100 mM sodium phosphate, pH 2.0 to 7.0; 100 mM Tris–HCl pH 8.0 to 12) and preincubated for 5 min at

25°C before the addition of 50 μL of 2% azocasein. After 20 min of incubation with azocasein, the reaction was stopped by adding 400 μL of 20% TCA. The sample was placed on ice for 10 min and centrifuged for 5 min at 16,500×g. A total of 600 μL of supernatant was then mixed with 600 μL of

0.1 M NaOH. The absorbance at 420 nm was determined. Blanks (test tubes without enzyme extract) were run in all cases.

4.2.4 Effect of temperature on total proteolytic activity

The effect of temperature on total proteolytic activity was evaluated according to Zibaee et al.,

(2012), with modifications. Briefly, 170 μL of 0.1 M Tris buffer pH 8.0 and 50 μL of enzyme extract were preincubated for 5 min at 4, 10, 20, 30, 40, 50, 60, 70, and 80°C before the addition of 50

μL 2% azocasein. The assays were performed as earlier described.

4.2.5 Specific proteolytic activity

72

To determine trypsin-like and chymotrypsin-like activities, BApNA (Nα-benzoyl-L-arginine-p- nitroanilide) and SApNA (N-succinyl-L-Ala-L-Ala-L-Pro-Phe-p-nitroanilide) were used. Trypsin-like activity was assayed according to Zibaee et al., (2012) using a final concentration of 1 mM BApNA.

A total of 50 μL of enzyme extract and 370 μL of Britton–Robinson buffer with the desired pH (3-

11) were pre-incubated for 5 min at 40°C, and 50 μL of BApNA was then added and the mixture incubated for 20 min at 40°C. The reaction was stopped with 500 μL of 30% acetic acid, and the tubes were centrifuged for 5 min at 16,500×g. The absorbance was measured at 410 nm.

Chymotrypsin-like activity was determined according to Zibaee et al., (2012), with modifications. A total of 50 μL of enzyme extract and 170 μL of Britton–Robinson buffer with the desired pH (6–11) were pre-incubated for 5 min at 40°C, and 50 μL of SApNA was then added and incubated for 20 min at 40°C. The reaction was stopped with 500 μL of 30% acetic acid, and the tubes were centrifuged for 5 min at 16,500×g. Absorbance was measured at 410 nm. Assays were conducted in triplicate, and appropriate blanks were run in all cases.

4.2.6 Enzyme kinetics

Kinetic studies for trypsin- and chymotrypsin-like serine proteases were performed according to

Hosseininaveh et al., (2009). For this study, five BApNA and SAp NA substrate concentrations

(0.01–0.09 mM) were evaluated. The Michaelis–Menten constant (Km) was evaluated with a regression analysis using the non-linear software GraphPad Prism (http://www.graphpad.com).

4.2.7 Inhibition assays

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4.2.7.1 In vitro assays. The proteolytic activity of enzymes present in the midgut of A. superciliosus was subjected to the effects of general and specific inhibitors. Soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), N-tosyl-L-phe- nylalanine chloromethyl ketone (TPCK), and iodoacetamide (IA) were evaluated according to Zibaee et al., (2012) at 0.01 and 0.1 mΜ using

BApNA or SApNA as substrates. A total of 20 μL of inhibitor, 40 μL of gut enzyme extract, and 380 μL of buffer were mixed and preincubated for 5 min at 40°C, and 40 μL of substrate was added to the mixture and incubated at 40°C for 20 min. The reaction was stopped by adding 600 μL of acetic acid (30%). The tubes were centrifuged at 16,500×g for 4 min, and the absorbance was measured at 410 nm. The assays were performed in triplicate, and test tubes without gut enzyme extract and inhibitor were used as a target and control in all cases.

4.2.7.2 In vivo assays. Insects were kept isolated without food for at least 48 h on moist filter paper in petri dishes at 25°C prior to the bioassays. The bioassays were performed according to the methodology described by Quintana et al., (2011). The maintenance diet consisted of leaves of

Maitenus boaria.

Insect mortality was evaluated according to Rebolledo et al., (2012). A total of 220 adult weevils

(110 males and 110 females) were placed individually in transparent plastic containers with fresh

Rubus ulmifolius leaves. A leaf that had previously been treated with inhibitors was placed inside each container, and an adult was added immediately. Solutions of each of the inhibitors PMSF,

TPCK, and SBTI with concentrations of 0.1, 1, and 10 mM were prepared in water or ethanol according to solubility. The control consisted of a leaf treated with the corresponding solvent (i.e., water or ethanol). Ten repetitions per sex and per each treatment were used. Every 24 h, the food (a fresh

R. ulmifolius leaf) was renewed after the appropriate inhibitor solution had been applied to the new

74 leaf. The number of dead insects was recorded after 24 h in contact with the treated leaf. This procedure was repeated at 48, 72, 96, and 120 h. Once the assay was finished, the average number of dead insects per treatment was determined. In addition, the weight of insects was recorded daily.

The data obtained were analyzed with a Fisher test (p < 0.05) using the statistical software StatsDirect v.2.7.8. To eliminate insect mortality resulting from causes unrelated to the treatment, mortality was corrected using Abbott’s formula (Silva et al., 2003).

4.3 Results

The hydrolysis of azocasein, a general proteinase substrate, by midgut extracts from A. superciliosus reached its maximum activity at pH 8.0, indicating that the activity was primarily due to alkaline proteases (Fig 3.1a). The hydrolysis of more specific substrates, BApNA and SApNA, provided evidence for alkaline proteinases (Fig 3.1b, c). BApNA was hydrolyzed maximally at pH 10, whereas the maximal hydrolysis of SApNA was at pH 11. Azocasein was hydrolyzed over a broad range of temperatures (20–50°C) and showed a maximum at 40°C (Fig 2). This value is very close to the temperature recorded for the maximum activity of serine protease in other insects (Tsybina et al.,

2005, Zibaee et al., 2012).

The total proteolytic activity of midgut extracts from A. superciliosus was characterized using the specific inhibitors PMSF (general serine proteinase inhibitor), SBTI (trypsin inhibitor), TPCK

(chymotrypsin inhibitor), and I A (cysteine proteinase inhibitor) and compared with the activity in the absence of inhibitors (Table 3.1). No inhibition was obtained when IA was used. The most effective inhibitor was SBTI suggesting that the proteolytic activity of the midgut of A. superciliosus is primarily tryptic serine protease activity.

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Fig. 4.1. Effect of pH on the hydrolysis of a azocasein, b BApNA, and c SApNA by midgut extracts from Aegorhinus superciliosus. The figure shows the mean ± standard error (n =3). Bars represent SE.

Kinetic studies indicated that the Km value of trypsin determined for the BApNA substrate was

0.28 ± 0.07 mM, whereas the Km value of chymotrypsin determined for the SApNA substrate was

0.38 ± 0.02 mM. These values are similar to those reported in other insects (Hosseininaveh et al

2009, Zibaee et al 2012).

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Fig. 4.2. Effect of temperature on the hydrolysis of azocasein by midgut extracts from Aegorhinus superciliosus. The figure shows the mean ± standard error (n =3). Bars represent SE.

Table 4.1. Effect of inhibitors on proteolytic activity of midgut extracts from Aegorhinus superciliosus using BApNA and SApNA as substrate.

To compare the efficacy of inhibitors in vivo, the specific inhibitors PMSF, TPCK, and SBTI

77 were incorporated into the diet. Mortality assays indicated that only SBTI caused death (Table

3.2). SBTI incorporated in the diet at 0.1 mM caused 100% mortality of females and 80% mortality of males of A. superciliosus, whereas higher concentrations of 1 and 10 mM caused the weight of the insects to decrease significantly (loss of weight of 50 mg in relation to the control, p < 0.05) as the result of an antifeedant effect.

Table 4.2. Effects of inhibitors on percent mortality of adult Aegorhinus superciliosus.

Inhibitor Insect sex Concentration (mM)

0.1 1.0 10

PMSF Males 0 0 0 Females 0 0 0

SBTI Males 80* 90* 50* Females 100* 80* 90*

Males 0 0 0 TPCK Females 0 10 0

Asterisk indicates significant difference in percent mortality between the treatment and the control

(Fisher test, p < 0.05).

4.4 Discussion

In Coleoptera, the midgut is the principal source of digestive enzymes and also one of the principal

78 sites for the absorption of digested material (Vásquez-Arista et al., 1999).

The maximum activity obtained at a pH range of 10–11 for trypsin and chymotrypsin is consistent with the results obtained by Oppert et al., (2003) for Tribolium castaneum indicating that the maximum hydrolysis of BApNA occurred at an alkaline pH range (8.5–9.2), whereas the maximum hydrolysis of SApNA as a specific substrate occurred at a pH range of 9.2–10.6. These pH ranges for maximum hydrolysis indicated the presence of trypsin and chymotrypsin, respectively. Furthermore, an optimal pH range of 7.0 to 10.0 for trypsin has been reported for many insects from different orders (Gooding and Huang 1969, Tong et al., 1981, Christeller et al., 1989, Hosseininaveh et al.,

2009), whereas an optimal alkaline pH range for chymotrypsin of 8.2 to 10.0 has been reported for insects belonging to the orders Orthoptera, Coleoptera, Diptera, Hymenoptera, and Lepidoptera

(Terra et al., 1996).

The optimum temperature reported is similar to that reported for other alkaline proteinases of insects (Gooding and Huang 1969, Pritchett et al., 1981, Zibaee et al., 2012), and the progressive loss of proteolytic activity observed in A. superciliosus above 40°C has also been recorded for proteases in other insect species (Bonete et al., 1984, Blanco-Labra et al., 1996, Pritchett et al.,

1981).

Proteolytic activity was strongly temperature dependent. The temperature range for optimal activity is consistent with those occurring in field during December–February, when daily maximums may exceed 30°C (Red agroclima 2014), while A. superciliosus is still active in Chilean berry fields.

Zibaee et al., (2012) reported that the high activity of enzymes at a specific temperature in in vitro assays reflects the temperature of the environment in which the organism feeds on its hosts.

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Extremes in temperature can cause an imbalance of different noncovalent forces (hydrogen bonds, ionic and Van der Waals interactions), leading to the denaturation of enzymes (Klibanov 1983).

A kinetic analysis was used to find Km values for trypsin and chymotrypsin activity. A low value of

Km would indicate strong binding between enzyme and substrate (Zibaee et al., 2012). Our results indicate a Km value of 0.28 mM for trypsin using BApNA as substrate and 0.38 mM for chymotrypsin using SApNA as substrate. A similar result was reported by Lam et al., (2000) for anionic trypsin (TRY2A) from the midgut of Locusta migratoria (Linnaeus) (0.26 mM). According to

Tsybina et al., (2005), the Km values for another trypsin-like protease, using BApNA as the substrate, ranged from 0.08 to 0.93 mM. Km values of chymotrypsin from insects, using SApNA as the substrate, ranging from 0.12 to 1.59 mM have been reported from the midgut of insects (Elpidina et al., 2005, Özgür et al., 2009).

Protein inhibitors were tested against midgut extracts from A. superciliosus. Protease activity was significantly reduced by SBTI at 0.1 mM, indicating primarily the presence of trypsin-like serine protease (76.8% inhibition) and, in a lesser proportion, of chymotrypsin-like serine protease (36.7% inhibition using TPCK at 0.1 mM). In contrast, cysteine protease did not contribute to total proteolytic activity. A similar result has been obtained by Purcell et al., (1992) in the boll weevil, Anthonomus grandis (Boheman), and three lepidopteran species. In these insects, soybean trypsin inhibitor inhibits protease activities by 63–72%. Christeller et al., (1989) have reported that SBTI totally inhibits BApNA hydrolysis in a crude extract of midgut protease from Costelytra zealandica (White), whereas a study by Sharifi et al., (2012) has shown 67.9% inhibition of midgut protease activity in

Osphranteria coerulescens (Redtenbacher) larvae using 1 mM of specific inhibitor.

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In the current study, it was found that a serine protease inhibitor (SBTI) incorporated in the diet caused a high percentage of mortality, indicating that trypsin-like proteases are vital for the growth and survival of the raspberry weevil, A. superciliosus. SBTI has previously been shown to be toxic to insects from different orders (Dorrah 2004, Franco et al., 2004, Silva et al., 2006, Saadati &

Bandani 2011). Therefore, blocking the activity of digestive trypsin using transgenic plants could be an alternative for the management of the raspberry weevil.

In conclusion, this study shows the predominance and importance of trypsin- like protease in the midgut of A. superciliosus. The control of this insect could target specific inhibitors of trypsin- like protease.

As the principal damage to the host plants is caused by the activity of root-feeding larvae, which can kill the plants, future work should be focused on the evaluation of the importance of trypsin- like protease in A. superciliosus larvae as well as the evaluation of the adaptive response of this insect to proteinase inhibitors.

4.5 Acknowledgments. The authors thank The National Commission for Scientific and Technological

Development (Conicyt) through Fondecyt Project #1100812 for its financial support of this research.

V. Medel thanks The National Commission for Scientific and Technological Development (Conicyt) for thesis support through scholarship 24090126.

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CHAPTER V

General discussion and concluding remarks

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5.1. General Discussion

5.1.1 Chapter II

Morphology Digestive System

The coleopteran food channel has been described by numerous researchers, providing background information on the internal structure of several families. In this study, the morphological and histological characterization of the digestive system was performed for the first time in A. superciliosus (Coleoptera: Curculionidae), a pest insect on berries, native to the southern cone of

America (Chile and Argentina). To carry out this characterization, approximately 60 complete intestines were extracted in males and 60 in females, and those that were completely inactive were used. The results of this investigation will allow to compare the histological samples made before and after feeding insects with protease inhibitors, and thus be able to complete objective II.

The digestive tract of insects is a continuous tube that extends from the mouth to the anus and passes through different regions containing valves and sphincters. Morphologically, the digestive tract in Aegorhinus superciliosus is divided into foregut (stomodeum), midgut (mesenteron, stomach) and hindgut (proctodeum), with the foregut and hindgut being of ectodermal origin, and the midgut of endodermal origin, which is consistent with reports by Borror and De Long (1969),

Belkin (1976), Snodgrass (1993), Caldeira et al., (2007); Bu and Chen, (2009), Chapman (1998;

2013), De Sousa et al., (2017) and Terra and Ferreira (2003; 2020) for other insects.

In the raspberry weevil, the foregut occupies 5%, the midgut 50% and the hindgut 45% of the total

83 length in the female. In the case of the male, the respective percentages were 7, 50 and 43% of the total length of the digestive tract. In this case, the three regions adjust to the proportions presented by Calder (1989), who carried out the evaluation of the proportion of the three regions of the digestive tract to various species of Curculionidae. The results obtained are similar to those reported in Rhynchophorus palmarum (Sánchez et al., 2000), Anthonomus grandis (MacGown and

Sikorowki 1981) and in Hypera postica (Tombes and Marganian 1967), but differ somewhat from those reported by Rubio et al., (2007) for Hypothenemus hampei, since the stomodeum occupies around 16%, the mesenteron 44% and the proctodeum 40%, therefore these values are comparable with those recorded by Díaz et al., (1998) in species of the genus Dendroctonus (Curculionidae:

Scolytinae), which present proportions of the stomodeum between 15 and 25%, the mesenteron between 42 and 52% and the proctodeum between 30 and 40% and with results obtained by Bu and Chen, (2009) for Dendroctonus armandi in which the anterior intestine occupies 18.7%, the middle intestine comprises 46.6% and the posterior intestine 35.9%.

The length of the organs that are part of the digestive system, in A. superciliosus, was compared to corroborate if there are significant differences in their size, according to sex. To carry out this study, the t Student test was applied. The results indicate that the female's pharynx and esophagus extent 1.21 mm. and 1.72 mm in the male, according to this test, there are significant differences in the indicated values, which suggests that in the male these organs as a whole are of greater length. Similarly, in the proventriculus the measurements were 0.94 mm. and 0.88 mm. for females and males respectively, therefore there are no significant differences between these values. In the case of the anterior ventricle, the female measured 8.88 mm. and, in the male, 8.10 mm. According to this there are significant differences between both sexes, being longer in the female.

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Agree to the observed, the female's posterior ventricle measures 11.34 mm and 10.5 mm in the male, therefore there are significant differences between both values, which means that this organ in the female would be longer. In the Ileon the values are 6.54 mm and 6.38 mm, for females and males respectively, so there are no significant differences between the two lengths. The colon, according to the measurements made, is 3.36 mm. length in the female and 2.46 mm. in the male, indicating that there are significant differences in the length of this portion. In the case of the rectum

5.75 mm. it is the length in the female and 4.32 mm. in the male, then there are significant differences between the two values, being longer in the female. Finally, the anus measures 2.65 mm. in the female and 2.92 mm. in the male, there are no significant differences between both lengths. These results indicate that, in the majority of the organs, there are differences in the length, for which it could be concluded that in the female the length of the digestive system is greater.

There are no reports comparing the length of these organs in other insects.

In curculionids, the midgut begins at the stomodeal valve, differentiates into anterior midgut and posterior midgut, and terminates at the pyloric valve (Baker et al., 1984; Sánchez et al., 2000;

Rubio et al., 2008; Bu and Chen, 2009). The insertion of the Malpighi tubes into the alimentary canal marks the beginning of the posterior intestine. There are six Malpighi tubes separated into two groups, the first group consisting of four long tubules extended forward, and the second group consisting of two tubules directed backwards. The above coincides with that reported for R. palmarum (Sánchez et al., 2000), C. sordidus, M. hemipterus and M. hebetatus (Rubio and Acuña,

2006), Compsus sp. (Rubio and Acuña, 2007), H. hampei (Rubio et al., 2007; 2008), D. armandi

(Bu and Chen, 2009). Furthermore, it coincides with that reported by Wigglesworth (1974),

Crowson (1981), Calder (1989) and Snodgrass (1993) who have recorded between four and six

Malpighi tubes in other beetles studied.

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Macgown and Sikorowski (1981) reported that in A. grandis the posterior ventricle is externally lined by gastric caeca, in this case A. superciliosus has some similarity with A. grandis and D. frontalis Zimmerman, 1868 (Díaz et al., 2000) , since these authors point out that several gastric caeca were identified in the terminal portion of the posterior ventricle, which coincides with our results, as well as with what was reported by De Sousa and Conte, (2013) for Sitophilus zeamais, contrary case observed in R. palmarum because according to Sánchez et al., (2000) these would be two and would be found in the anterior ventricle. Furthermore, Rubio and Acuña (2006) observed that the gastric caeca of C. sordidus, M. hemipterus and M. hebetatus, were found in both the anterior and posterior ventricles, which is in agreement with that reported by Baker et al., (1984) for S. granarius, which differs from our results and the reports of other authors.

The anatomical organization of the digestive tract of this insect is very similar to that of other coleoptera, although there are some important variations such as crop and rectal pads absence and peritrophic membrane presence. Our results agree with those reported by Sanchez et al., (2000) for R. palmarum, but differ from a report by Rubio and Acuña (2006) for C. sordidus, M. hemipterus and M. hebetatussince, Rubio et al., (2007) for Hypothenemus hampei, Omotoso,

(2013) for Rhynchophorus phoenicis because in these insects have been identified the structures mentioned above.

Histology Digestive System

The foregut is made up of a layer of circular muscles, followed by a thick layer of longitudinal muscles, which is continued in a simple flat epithelium, which coincides with that reported by

Omotoso, (2013) for R. phoenicis. Internally, a simple cylindrical epithelium is observed, whose

86 cells have an ovoid nucleus at the same level in the basal region. Finally, and in direct contact with the lumen, the intimate layer is observed, a thin, dense, amorphous layer folded towards the interior and to the muscular epithelium.

The anterior ventricle has columnar cell epithelium with a big, ovoid central nucleus and can form small folds, similar to that found in P. anxia (Berberet and Helms 1972), R. palmarum (Sánchez et al., 2000), T. collaris (Wanderley-Teixeira et al., 2006), D. armandi (Bu and Chen, 2009), R. phoenicis (Omotoso, 2013). In addition, it presents an internal border covered by microvilli, characteristics, also described by Bu and Chen, (2009) for D. armandi, Omotoso, (2013) for R. phoenicis, Snodgrass (1993) and Chapman (1998) for insects in general.

In A. superciliosus, the lumen or cavity through which the digested food circulates, is formed by a thin layer of epithelial cells called the “peritrophic membrane” that is highly permeable. Brandt et al., (1978), Eisemann and Binnington (1994), Jarial and Engstrom (1997), Terra (1990), Bolognesi et al., (2008), Toro et al., (2009), Terra et al., (2006; 2019) and Terra and Ferreira, (2020), explain that it is an anatomical structure that surrounds the food bolus in beetles and in most insects, since it allows the passage of gastric secretion that will penetrate the food mass. Furthermore, this membrane, according to Crowson (1981), would be related to food intake, since it would only exist in beetles that consume foods with high solids content. These results coincide with those reported by Vásquez-Arista et al., (2001) for Prostephanus truncatus (Horn 1878), but they differ from those reported by Sánchez et al., (2000) for R. palmarum, Berberet and Helms (1972) for P. anxia, and Chadbourne (1961) for A. grandis since this membrane is not present in these insects.

In the case of the proctodeum, it is made up of a layer of circular and longitudinal muscles that

87 shape and participate in the operation of this region, as well as presenting undulations and invaginating folds. These observations are similar to those described by Berberet and Helms,

(1972) for P. anxia, Bu and Chen, (2009) for D. armandi and Crowson (1981) for beetles in general.

It is also made up of the ileum, a long, thin tube, similar to that described for D. adjuntus Blandford,

1897 (Zuñiga et al., 1994) and for D. armandi (Bu and Chen, 2009), but different from that found in other beetles, where it is described as short or as an undifferentiated organ (Chapman 1998).

The colon is externally lined by a wall of muscle cells and presents in its internal part undulations and folds invaginated with the presence of villi, which agrees with what was reported by Sánchez et al., (2000) for R. palmarum. The portion of the colon that is close to the rectum does not have villi, the rectum being a large, thin-walled sac (rectal ampulla) that has a cylindrical epithelium of muscle cells with contractile capacity, with no rectal pads. Similar characteristics were observed by Omotoso (2013) for R. phoenicis. Snodgrass (1993) explains that in some beetles these organs would be absent, differing from that reported by Zuñiga et al., (1994) for D. adjuntus.

Finally, there is the anus, a short tube which is attached to the reproductive system, which coincides with that reported by Wigglesworth, 1974; Crowson, 1981; Sánchez et al., 2000; Rubio and Acuña,

2006, for other beetles.

5.1.2 Chapter III

Effect of protease inhibitors on the digestive system

In the present study, the histopathological effects of protease inhibitors on the digestive system

88 tissue of the adult insect A. superciliosus are evaluated. Studies by other researchers on the effects that PI cause on the tissue of insects fed with these compounds, are scarce and have been carried out on larvae of different stages and not on adults. In these investigations, the effect of Bacillus thuringiensis toxin on the intestine of insect pest larvae is evaluated. Despite not coinciding with the state of development of the insects evaluated in the aforementioned studies, these results were compared with those obtained in the present investigation.

The results indicate that protease inhibitors do not exert any negative effect on the tissue of the digestive system of the adult insect. This is because the histological samples made after feeding the insects with PIs have morphological characteristics similar to those observed in the histological samples made before feeding the insects with these compounds. Features described in Chapter II.

These results do not agree with other investigations developed to evaluate the damage caused by protease inhibitors or other compounds to the tissue of the digestive system of Aedes aegypti,

Anopheles albimanus, Culex quinquefasciatus, Ostrinia nubilalis and Podisus nigrispinus. The main alterations observed in these insects were partial or total cell destruction, high cytoplasmic vacuolization, and increased ectoperitrophic space. In addition, damage was observed in the midgut and in the gastric caecum in dead larvae that were treated with these compounds, since they presented the disorganized epithelial layer, most of the missing cells and the destroyed peritrophic membrane (Lahkim -Tsror, et al ., 1983; Denolf et al., 1993; Arruda et al., 2003; Ruiz et al., 2004;

Kabir et al., 2013; Sasaki et al., 2015; Dos Santos Junior et al., 2020). In the case of A. superciliosus, none of these damages was observed, since the samples showed a tissue with complete circular and longitudinal muscles, cells with visible nuclei, without cell lysis

(characteristics presented in Chapter II).

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Among the histopathological changes that could have occurred in the morphology of the cells present in the midgut of the adults of A. superciliosus, fed with blackberry leaves treated with PIs, the following stand out: completely destroyed cells, without visualizing the limits of these, which would indicate the harmful action of PIs. Cells of the middle intestine filled with spaces, and the apical part with poorly developed microvilli. In addition, destruction of the mitochondria, characterized by the loss of ridges and changes in content; thickening or destruction of the peritrophic membrane, with the consequent increase in the sub-peritrophic space, destruction of the cytoplasm, which is characterized by the destruction of vacuoles, absence of organelles and decrease in the size of the microvilli.

Antifeedant effect of protease inhibitors.

The antifeedant effect of protease inhibitors on A. superciliosus was evaluated. The results show that males and females consumed the leaves previously sprayed with inhibitors. According to the

Kruskal Wallis test, there are no statistically significant differences between control (water) and

SBTI treatment (P ≥ 0.05) in the area of average consumption.

Regarding the PMSF and TPCK inhibitors (p ≤ 0.05), it was concluded that there are significant differences between these treatments and the control (ethanol). This because the leaf area consumed by the insects fed with PMSF and TPCK, was less than the area consumed by the insects in the control (ethanol). This indicates that these inhibitors would have an anti-alimentary effect on the adult raspberry weevil.

On the other hand, the SBTI inhibitor did not produce any anti-alimentary effect, since both males

90 and females were fed in the same way as the control individuals. In other studies, has been tested as an anti-nutritional factor in the metabolism and development of some insects, specially in larvae feed artificially with SBTI, such as: the European borer Ostrinia nubilalis (Larocque and

Houseman, 1990), the soldier beetworm Spodoptera litura (McManus and Burgess, 1995) the tobacco heartworm Heliothis virescens (Johnston et al., 1995), the melon fly Bactrocera cucurbitae

(Kaur et al., 2009), this inhibitor significantly delayed the larval period, delayed pupation and the remaining development period compared to the control. This delay was directly proportional with the increase in the SBTI concentration. These studies suggest that the severe retardation in growth and development caused by the inhibitor would provide a much longer period in the development of the insect until the appearance of adults. Furthermore, it has been shown to be effective against proteases of the midgut of the flour beetle, Tenebrio molitor (Applebaum et al., 1964), the trypsin of the horned tobacco worm, Manduca sexta (Miller et al., 1974; Shukle and Murdock, 1983), and the proteases of the field black cricket Teleogryllus commodus (Burgess et al., 1994). In this case, the results are even more promising, since the insects fed with SBTI consumed the food in a normal way, finally causing death, both males and females, producing in the latter the highest percentage of mortality.

Insecticidal effect of protease inhibitors.

When evaluating the effect of protease inhibitors on artificially fed insects, the results show that there is a relationship between increasing concentrations and the number of dead specimens, due to the effect exerted by PMSF and TPCK inhibitors. However, considering the criteria proposed by Silva et al., (2002) that indicate that only treatments with a mortality greater than 40% are promising, these inhibitors would not be effective in causing the death of the insect.

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According to the above, it was observed that the SBTI inhibitor registered a higher percentage of mortality and efficiency after the third application in all the concentrations tested. When evaluating the cumulative mortality at 120 hours, the T3 treatment (SBTI) reached 100% mortality, while the

PMSF and TPCK inhibitors showed little effectiveness in all the concentrations tested, producing only 10% mortality in the individuals. Therefore, there are statistically significant differences between the applied treatments and their respective controls. If we compare mortality between males and females, the results indicate that in insects fed with SBTI, death occurs mainly in females. However, in those fed PMSF and TPCK, mortality occurs mainly in males. These results cannot be compared with other studies, since the effects of these inhibitors on insects have not been evaluated in each sex separately.

In an investigation by Ortego et al., (1998), these authors tested several serine protease inhibitors

“in vivo” in larvae and adults of A. mariaefranciscae, the results showed that survival was strongly reduced and development was significantly delayed in larvae fed an artificial diet containing SBTI.

The mortality rates obtained represent an increase of 35-45% with respect to the mortality of the controls, and are among the highest percentages reported so far on curculionids. However, the results obtained in this study far exceed the percentage obtained in the research by Ortego et al.,

(1998), since the mortality of the raspberry weevil reached 100% in the females fed with SBTI, which it is a promising result for future control strategies.

On the other hand, Purcell et al., (1992) artificially fed the cotton weevil with high levels of SBBI and SBTI, and in their results they found no mortality or growth retardation of the insect. However,

Graham et al., (2002) observed that the cowpea trypsin inhibitor in strawberry is effective to protect the roots against the feeding of vine weevil larvae. Similarly, Zhao et al., (2019) indicate that SBTI

92 negatively affects Plutella xylostella larvae and adults, delaying the growth and development of these states, but does not cause the death of the insect.

5.1.3 Chapter IV

Effect of pH on total proteolytic activity

Digestion is a phase of insect physiology in which surprisingly in Chile, little research has been done, considering the importance of food for insects and the fact that the most important control measures involve the action of pesticides that directly affect the digestive enzymes of pests.

In Coleoptera, the midgut is the principal source of digestive enzymes and also one of the principal sites for the absorption of digested material (Vázquez-Arista et al., 1999; Terra and Ferreira, 2020).

The enzymatic extract used for the inhibition tests was obtained according to the methodology of

Pereira et al., (2005). For this, the insects were frozen at -18 ° C, and the midgut of 80 insects was removed and placed on ice.

Studies show that the total proteolytic activity present in the digestive tract of A. superciliosus has a maximum activity at pH 8.0, which would indicate the presence of mainly basic proteases and more specifically, the serine protease type. The alkaline pH of the midgut of this insect is similar to that reported in other species of the Curculionidae family (Adedire, 1990; Girard et al., 1998a;

Bonadé-Bottino et al., 1999) and for insects of the order Lepidoptera, where the optimal pH is in the alkaline range (Pritchett et al., 1981; Terra, 1988; Purcell et al., 1992; Lee and Anstee, 1995;

Abo-El-Saad, 2000).

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Effect of temperature on total proteolytic activity

Optimal temperature results for enzyme activity show a maximum at 40 ° C. This value is very close to that recorded for the maximum activity of the serine protease extracted from Trichoplusia ni, which occurs at 45 ° C (Pritchett, 1981). The progressive loss of proteolytic activity observed in A. superciliosus above 40 ° C has also been reported for proteases from M. auratus (Bonete et al., 1984), Tribolium castaneum (Blanco- Labra et al., 1996) and Trichoplusia ni (Pritchett, 1981), which also indicates the thermal instability of the enzyme. In general, the proteolytic activity presents good thermal stability in the range between 30 and 50 ° C, reaching within this range relative activity values greater than 60%.

Effect of pH on the Trypsin and Chymotrypsin activity

According to the results obtained, it is possible to appreciate a greater enzymatic activity at pH 10 and 11 using BApNA and SApNA as specific substrates respectively, which would be confirming the presence of trypsin and chymotrypsin in the intestine of A. superciliosus. The maximum activity obtained at pH 10 for trypsin, using BApNA as a specific substrate, agrees with the results obtained by Oppert et al., (2003) for T. castaneum, who indicate that the maximum hydrolysis of BApNA occurred in an alkaline pH range (8.5-9.2) thus detecting the presence of trypsin. Furthermore, they indicate that the maximum hydrolysis of SApNA as a specific substrate occurred in the pH range

9.2–10.6, which indicates the presence of chymotrypsin, similar results were obtained in A. superciliosus whose optimal pH for the same enzyme was 11.

Many investigations carried out on insects indicate that trypsin is the most common protease and

94 the one with the highest activity within total proteolytic activity (Baker, 1977), whereas chymotrypsin is a protease that can (Gooding, 1975) or not be present (Yang and Davies, 1971).

Similarly, Al Jabr and Abo-El-Saad, (2008) reported that in R. ferrugineus the optimum for trypsin was observed at pH 9.5. This estimate is consistent with other results obtained by Peterson et al.,

(1994) and Abo-El-Saad (2000), who reported an optimal pH of 10.5 and 10 for trypsin activity in

Manduca sexta and Spodoptera littoralis respectively. Likewise, Johnston et al., (1995) reported an optimum of pH 10 and 11, for Heliothis virescens, indicating that it would be trypsin and chymotrypsin respectively.

In Spodoptera littoralis larvae, Marchetti et al., (1998) found trypsin with optimal activity at pH

10. Similarly, in cotton boll weevil (Anthonomus grandis), serine proteases appear to be the main enzymes in the midgut (Purcell et al., 1992). In Trogoderma granarium, wheat plague, serine proteases, trypsin and chymotrypsin have been reported as the major endopeptidases in the extract of the midgut of this insect (Hosseininaveh et al., 2007).

Enzyme kinetics

Km is a measure of the stability of the enzyme-substrate complex, and a low Km value may indicate strong binding between both compounds (Zibaee et al., 2012). In this investigation, the

Km value of trypsin using BApNA as specific substrate was 0.28 ± 0.07 mM. This result is comparable with the reported Km values for trypsin isolated from the midgut of various insect species: 0.121 mM in Musca domestica (Lemos and Terra, 1991), 0.41 mM in Ostrinia nubilalis larvae (Bernardi et al., 1996), 0.37 mM in Costelytra zealandica (Christeller et al., 1989), 0.26 and

95

0.47 mM for trypsin isoforms in Locusta migratoria (Lam et al., 2000), 0.69 ± 0 , 01 mM in

Osphranteria coerulescens (Sharifi et al., 2012) and a Km value of 0.6 mM in Eurygaster integriceps (Hosseininaveh et al., 2009), using the same substrate. In turn, the Km value for chymotrypsin using SApNA as specific substrate was 0.3866 ± 0.02111mM.

According to the result of Km for trypsin using BApNA as specific substrate, the value 0.28 ± 0.07 mM, is one of the lowest values reported in enzyme inhibition studies in insects, which indicates that there is a strong affinity of this enzyme by the substrate.

In vitro assays.

The inhibition effect on enzyme activity was evaluated for the following inhibitors: PMSF, SBTI,

TPCK and IA. The concentrations evaluated were those described by Zibaee et al., (2012). This is because they are used by researchers in most investigations of this type. The results obtained indicate that SBTI at 0.1 mM achieves 76.8% inhibition using BApNA as a specific substrate, which would indicate a greater presence of trypsin as a digestive enzyme. This agrees with the results obtained by Alarcón et al., (2002) for R. ferrugineus who obtained a 78% inhibition at a concentration of 12 mM. for the same inhibitor. Similarly, Al Jabr and Abo-El-Saad, (2008) used

SBTI as a specific trypsin inhibitor for digestive extracts of R. ferrugineus, reaching an inhibition of 70% and 100% when amounts of 15 µg and 25 µg were applied respectively. In turn, Dorrah

(2004) indicated that when using SBTI as an inhibitor on trypsin and chymotrypsin found in S. littoralis larvae, the results showed that SBTI is an effective inhibitor of these enzymes, explaining that the enzymatic activity was inhibited by 92 and 98% at concentrations of 2.0 and 5.0% of SBTI

96 respectively and that at lower concentrations of the inhibitor (0.1, 0.2, and 0.5%) the activity was inhibited in 27, 47 and 75 %, respectively.

Similar results were obtained by Purcell et al., (1992) in Anthonomus grandis and three species of lepidoptera. In these insects, SBTI inhibited protease activities between 63 and 72%. Similarly, a study by Sharifi et al., (2012) showed a 67.9% inhibition in intestinal protease activity in

Osphranteria coerulescens larvae using 1 mM of specific inhibitor.

For its part, TPCK achieves an inhibition percentage of 36.7% using SApNA as a specific substrate.

These results differ from that reported by Alarcón et al., (2002) for R. ferrugineus where it reached

2.9% (concentration 5 mΜ) of inhibition, using TPCK, which is much lower than that observed in

A. superciliosus, where the inhibition reaches a maximum of 36.7%. For IA, no inhibition is observed, which differs from that found in R. ferrugineus reaching 3.3% (0.1 M concentration).

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5.2. Concluding remarks

In this research, various topics related to the raspberry weevil A. superciliosus, a pest of economic importance in berries, were addressed. Due to the lack of information regarding the morphology, histology and biochemistry of this insect, coupled with the ineffectiveness of the methods used to control it, it was decided to study these unknown, but valuable aspects when making decisions for the choice of strategies for control.

The morphology and histology of the digestive tract of the raspberry weevil Aegorhinus superciliosus (Coleoptera: Curculionidae) is described for the first time. The results obtained indicate that it presents the same organization and typical structures described for most insects and for other beetles of the same family. The female has a longer digestive tract than the male, which would be closely related to the reproductive function it performs. This insect presents notable differences such as: absence of crop, number and position of gastric blinds, presence of peritrophic membrane and absence of rectal pads. Histologically, it is made up of circular and longitudinal muscles, pseudostratified columnar epithelial tissue with four types of cells: columnar, goblet, regenerative, and endocrine cells. All of these characteristics are similar to those described for most insects.

According to the background presented and because there are no studies of the internal anatomy and histology of A. superciliosus in our country, this research is considered to represent the first contribution to the knowledge of basic aspects of this insect, since it is described anatomically the digestive system of the adult insect in which the fundamental digestive processes for the growth

98 and development of this pest are carried out.

Like humans, insects must obtain ten essential amino acids through the digestion of protein from food. Its digestive proteases catalyze the breakdown of proteins to generate the free amino acids necessary for their growth and development; these enzymes, therefore, are potential targets for pest control in agriculture. For this reason, this study allowed demonstrating that the total proteolytic activity present in the digestive tract of A. superciliosus shows a maximum of activity at pH 8.0, which would indicate the presence of mainly basic proteases and specifically of the serine protease type. Likewise, the results obtained indicate a higher specific enzymatic activity at pH 10 and 11, which confirms the presence of trypsin and chymotrypsin in the midgut of this insect. Furthermore, the maximum enzymatic activity was reached at an optimal temperature of 40 ° C.

Another important result was the one obtained in enzymatic kinetics, where the Km value of trypsin and the Km value of chymotrypsin were 0.28 ± 0.07 mM and 0.38 ± 0.021 mM respectively. Values that, according to other reports, would be considered, as excellent, especially the Km of trypsin, since this shows that the enzymes of the middle intestine of the raspberry weevil have a high affinity for the substrate used, which represents data important when analyzing the use of compounds aimed at affecting your diet.

When evaluating, the inhibition effect on the total enzymatic activity for the following inhibitors:

PMSF, SBTI, TPCK and IA. The results obtained indicated that the highest percentage of inhibition was caused by the specific inhibitor SBTI (76.8%), while the serine protease inhibitor PMSF achieves 82.7% inhibition; for its part, TPCK achieves only 36.7% inhibition. In the case of IA, no

99 inhibition was observed. These results suggest that the proteases present would be mainly of the serine protease type, and specifically trypsin and chymotrypsin, which is related to the results obtained in the "in vivo" studies.

Regarding the tests carried out “in vivo”, it was observed that the inhibitors PMSF and TPCK, achieve to cause an anti-alimentary effect in the insect, a rather promising result, since there are reports in which these compounds do not exert negative effects on the insects fed on them. In addition, the specific inhibitor SBTI, did not cause this same effect, but managed to kill most of the fed specimens, especially the females. This result is extremely positive, if you think it is the first study, related to the possible enzymatic vulnerability of this insect.

In addition, the histopathological effects of protease inhibitors on the tissue of the digestive system of the adult insect were evaluated. The results indicate that these compounds do not exert any negative effect on the tissue. These results lead us to think that the destructive effects that various compounds exerted on larvae artificially fed with them in other studies are closely related to the state of development of these specimens, when feeding them with inhibitors.

With the results obtained, it can be concluded that the proposed hypothesis is approved almost in its entirety, since two protease inhibitors produce an anti-alimentary effect on adult insects, which affects their growth and therefore their development, and one of them it was able to cause the mortality of all the female specimens fed with it. As for the damage to the tissue of the digestive system, this did not occur, which does not mean that the inhibitors are inefficient, since they are capable of causing death.

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The use of anti-food in pest management has several advantages over the classic synthetic pesticide, such as specificity of the target insect, limited residual activity and environmental impact, and the low probability of development of resistance. However, much interdisciplinary effort is needed to determine the chemical basis of insect-host associations, which can allow the exploitation of natural products, for the control of specific pests.

The information obtained is important to assess the real biochemical vulnerability of this insect to various inhibitors, allowing new control strategies to be proposed, since future research could focus on the search and evaluation of specific natural inhibitors. However, there are still many aspects that remain to be known if we intend to advance along this path. In our country, the biochemistry of insects and specifically the study of digestive proteases of insects and their respective inhibitors, is still an unknown topic, so this thesis is the beginning in the search for new control methodologies for this, and other insects, as many studies are necessary before a protease inhibitor or the appropriate combination of these can be recommended.

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