Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Acoustic recording as a non‐invasive method to detect larval infestation of Cosmopolites sordidus

The banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), is a major pest of banana cropping systems, as larvae bore into the corm of banana plants and damage the insertion points of primary roots, leading to plant snapping and toppling. Plant infestation by the banana weevil is assessed only via invasive techniques, such as plant cutting that highlights larval tunnels. We present here a non‐invasive technique based on a bioacoustic sensor with a band‐pass filter to detect larval activity inside banana corm. On the basis of a test under controlled conditions, we observed that the range of larval activity was between 1.4 and 2.6 kHz. Pulses lasted ca. 168 ms and were separated by intervals of 300 ms. The technique enables detection of more than 90% of infested suckers. The use of this technique to understand larval development of banana weevil is discussed.     

Preference of Cosmopolites sordidus for fusarium wilt-diseased banana plants

The objective of this study was to understand the role of Cosmopolites sordidus in the dispersal of Fusarium oxysporum f. sp. cubense race 1 (Foc) and more specifically to determine whether C. sordidus is preferentially attracted to Foc-contaminated banana plant material vs. Foc-free material. The attraction of C. sordidus to healthy and Foc-contaminated Gros Michel banana bulbs and pseudostems, as well as to pure cultures of Foc, was compared using four-choice olfactometers. Males and females were studied separately. After storage in a dark room, weevils were placed in the central chamber of olfactometers. After 15 min, the orientation of each of the weevil was determined according to the food source located in each of the four peripheral chambers of the olfactometer. Overall, 560 olfactometer assays were carried out. Weevils were found to prefer Foc-infected plant tissues. Weevil preference was not significantly affected by weevil sex. The attraction of C. sordidus to Foc-contaminated plant material suggests that this insect could be important for the dissemination of Foc, especially in early stages of epidemics, when Foc-infected plants are scarce.     

The challenge of biological control of Cosmopolites sordidus Germar (Col. Curculionidae): A review

The banana weevil (also known as banana root borer) Cosmopolites sordidus Germar (Coleoptera Curculionidae) is the major pest of banana and plantain. Because banana ranks 2nd in fruit production worldwide, this pest has substantial social and economic importance. The biological control of the C. sordidus remains challenging because of its behaviour and resistance to parasitism and predation. The last review concerning the biological control of C. sordidus was published two decades ago, and relevant knowledge and methods have developed in the interim. The present paper provides an update of that knowledge and summarizes past and current challenges as well as providing perspectives on achieving sustainable control of C. sordidus. We first discuss studies on the classical biological control of C. sordidus, underlining the limits of classical biological control methods such as the importation of predators, parasitoids or pathogens. Next, we consider conservation biological control of C. sordidus, with a focus on ants. We also highlight an ‘arthropod bias’ that has led to a lack of information on the role of vertebrates in the regulation of C. sordidus.     

Banana defence responses to Cosmopolites sordidus feeding and methyl jasmonate application

Each year 25–75% of banana and plantain yields are lost because of rhizome damages caused by banana weevil (Cosmopolites sordidus) in growing regions of sub‐Saharan Africa. However, the specific plant defence response of the rhizome tissue in relation to the C. sordidus attack is unknown. Consequently, in this study, we evaluated whether plant defence substances in the rhizome are correlated with the degree of larval damage and whether applications of methyl jasmonate (MJ) elicit a greater induction of the plant defence potential against C. sordidus. Moreover, we attempted to reveal cellular modifications in response to the root feeding herbivore through histochemical staining. The banana cultivars “Km5” and “Mbwazirume” with tolerance and susceptibility to C. sordidus, respectively, were used in a pot experiment to evaluate percent rhizome damage, leaf chlorophyll content, total phenolic content (TPC), antioxidant capacity and cell morphology in response to C. sordidus attack and/or MJ applications compared to untreated control plants. We found that C. sordidus‐induced rhizome damage was 30% in the susceptible cultivar but less than 5% in the tolerant cultivar. The percent rhizome damage was not related to leaf chlorophyll content but showed a significant negative linear relationship to both TPC and antioxidant capacity. Larvae feeding induced a considerably greater increase of polyphenolic defence compounds in Km5 than in Mbwazirume; however, this response was opposite in the MJ treatment, suggesting that the phytohormone induced the susceptible plant to invest more into the synthesis of defence chemicals that in turn lead to reduced C. sordidus damage. Tissue staining demonstrated a greater deposition of lignin and suberin in C. sordidus challenged rhizome, presumably to seal off healthy tissue with a physical barrier from continued pest attack. It is concluded that MJ induces polyphenolics in susceptible Mbwazirume banana that reduced C. sordidus damage.     

Different bioassays for investigating orientation responses of the banana weevil, Cosmopolites sordidus, show additive effects of host plant volatiles and a synthetic male-produced aggregation pheromone

Abstract Three different bioassay methods to investigate the orientation behaviour of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), to host plant volatiles and a synthetic pheromone (cosmolure+) were compared. A locomotion compensator was used to separately record walking tracks in response to three odour sources. The data show that C. sordidus uses odour‐conditioned anemotaxis in its orientation to the odour sources tested. Of the two olfactometers tested, a dual port olfactometer using a continuous airflow showed stronger discrimination by C. sordidus to the different odours compared with a double pitfall olfactometer. The results of all three bioassays indicate that C. sordidus responds in an additive way to the combination of fermentation plant volatiles and the synthetic pheromone.     

Dual inoculation of Fusarium oxysporum endophytes in banana: effect on plant colonization,growth and control of the root burrowing nematode and the banana weevil

The burrowing nematode (Radopholus similis (Cobb) Thorne) and the banana weevil (Cosmopolites sordidus Germar, Coleoptera: Curculionidae) are major pests of banana (Musa spp.) in the Lake Victoria basin region of Uganda. Among biological options to control the two pests is the use of non-pathogenic Fusarium oxysporum Schltdl.: Fries endophytes of banana. We investigated the ability of endophytic F. oxysporum isolates Emb2.4o and V5w2 to control the banana weevil and the burrowing nematode, alone and in combination. Plant colonization by the endophytes was determined by inoculating their chemical-resistant mutants separately and in combination, onto banana roots. Plant growth promotion was determined by measuring plant height, girth, number of live roots and fresh root weight at harvest, and control of the nematode and weevil was determined by challenging endophyte-inoculated plants with the pests 8 weeks after endophyte inoculation. Endophytic root colonization was highest in plants inoculated with both endophytes, compared with those inoculated with only one of the endophytes. Root colonization was better for isolate V5w2 than Emb2.4o. Dually inoculated plants showed a significant increase in height, girth, fresh root weight and number of functional roots following nematode challenge. Nematode numbers in roots were reduced 12 weeks after challenge of 8-week-old endophyte-inoculated plants. Significant reductions in weevil damage were observed in the rhizome periphery, inner and outer rhizomes, compared with endophyte non-inoculated controls. We conclude that dual inoculation of bananas with endophytic isolates Emb2.4o and V5w2 increases root colonization by the endophytes, reduces nematode numbers and weevil damage, and enhances plant growth in the presence of nematode infestation.     

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.     

Long-term impacts of elevated carbon dioxide and transgenic Bt cotton on performance and feeding of three generations of cotton bollworm

A field screening trial undertaken in Uganda showed that a number of Musa L. (Musaceae) cultivars and hybrids displayed high levels of resistance to banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), while most highland banana cultivars tended to be susceptible to weevil attack. In this study, research was undertaken to determine modalities of resistance to banana weevil. Laboratory studies suggested that all cultivars were attractive to the weevil and that females freely oviposited on all cultivars. While some differences were found between cultivars in attractivity and egg numbers, these were not related to subsequent damage. Therefore, antixenosis does not appear to play an important role in host‐plant resistance to banana weevil. Larval survivorship rates in living corms were, for the most part, low on resistant cultivars, suggesting that antibiosis mechanisms offer the primary avenues of resistance. In the laboratory, development was slower on some resistant cultivars although survivorship rates on excised corm material were not as well related to levels of resistance as that on living material. Sap appeared to play a minor role in reducing egg eclosion rates on some resistant cultivars. Methanol extracts from Kayinja, a resistant cultivar, inhibited larval development on corms of susceptible cultivars in the laboratory.     

Distribution, timing of attack, and oviposition of the banana weevil, Cosmopolites sordidus, on banana crop residues in Uganda

Crop sanitation (removal and chopping of residue corms and pseudostems following plant harvest) has been recommended as a ‘best bet’ means of reducing banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), populations. However, it has been unclear when such practices should be carried out and what types of residues should be destroyed. Therefore, trials were conducted in Uganda to determine C. sordidus distribution, timing of attack, and oviposition on crop residues and growing plants. Assessments were performed in on‐station trials on different aged standing and prostrate residues by destructive sampling. Similar data were collected from farmers’ fields maintained at low, moderate, and high levels of sanitation. In the on‐station trial, oviposition occurred on up to 120‐day‐old residues, although most occurred within 30 days of harvest. In a second on‐station experiment, oviposition on standing residues was not significantly affected by residue age. By contrast, oviposition on prostrate residues was two times higher on 4‐week‐old than on 2‐week‐old residues, while the number of larvae on 8‐week‐old residues was three times higher than on 2‐week‐old residues. The number of adults was twice as high on 16‐week‐old residues as that on 2‐week‐old residues for both prostrate and standing residues. Farmers’ fields maintained at high sanitation had 50% fewer eggs per residue than farms with low sanitation levels. In general, the number of immatures per residue was 50% higher on banana corms than on pseudostems. Numbers of larvae per residue were three times more abundant at low than at high sanitation levels. Residues in fields with high sanitation supported 50% fewer adults than residues in low sanitation fields. The results suggest that removal and splitting of corms after harvest is effective and practical in destroying immature growth stages of the pest and that such practices should be carried out soon after harvest.     

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy‐2 and Icy‐4 cystatin genes. Artificial microRNA lines independently silencing the two drought‐induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay‐green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy‐2 and Icy‐4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap‐1, HvPap‐12, and HvPap‐16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.     

Effect of fallows on population dynamics of Cosmopolites sordidus: toward integrated management of banana fields with pheromone mass trapping

• 1 The banana weevil Cosmopolites sordidus Germar (Coleoptera: Curculionidae) is the most serious pest of banana and plantain in most production areas, including the West Indies.
• 2 During a period of 2 years, we assessed the efficacy of a pheromone mass‐trapping control strategy of C. sordidus in field conditions in Guadeloupe at different cropping stages, both in sanitation fallows and in different ratoon banana crops.
• 3 In the fallows, catches peaked 3 months after beginning trapping and then decreased to zero after 9 months. By contrast, for the new plantations, the catches of C. sordidus increased after the 11th month and, in the older banana fields, there was no decrease in C. sordidus catches. The C. sordidus catches increased in the neighbouring banana plots, whereas they decreased in the fallows, and these catches decreased with the distance from fallow.
• 4 In conclusion, mass trapping with synergized pheromone traps within fallows should allow better sanitation of banana plantations. Yet, within the farms, fallows must not be located next to new plantations to avoid massive damage to the young plants. More generally, the present study emphasizes that the control of this insect should be managed at the farm scale and not at the field scale, with special attention being paid to the location of fallows.

     

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests.     

The emerging role of cystatins in Alzheimer's disease

Recently opposing effects of cysteine protease inhibitors, the human cystatins, on neurodegeneration have been reported. Human cystatin C is a risk factor for late‐onset Alzheimer's disease (AD), whereas human stefin B (cystatin B) has no direct involvement in AD. Conflicting data show that their target protease, cathepsin B, might be anti‐amyloidogenic, helping in amyloid‐beta (Aβ) clearance or, instead, might be involved in Aβ production. Some reports claim that cystatin C binds soluble Aβ, making transgenic animals healthier, others, in contrast, that deleting cystatins genes may contribute to amyloid pathology in animal models of AD.     

The use of aggregation pheromone to enhance dissemination of Beauveria bassiana for the control of the banana weevil in Uganda

Candidate strains of Beauveria bassiana were identified for use in integrated pest management of the banana weevil Cosmopolites sordidus. Horizontal field transmission of B. bassiana between banana weevils using different delivery systems, including aggregation pheromones, was investigated. We observed that infected weevils could transmit the fungal pathogen to healthy individuals. Most dead weevils (52%) due to B. bassiana infection were found at the base of banana plants in the leaf sheath or in the soil near banana plants. Significantly more weevils died from the pathogen in plots where B. bassiana was applied in combination with the pheromone than where it was applied alone. Our data demonstrate that C. sordidus aggregation pheromone can be a valuable tool to enhance the dissemination of B. bassiana for the control of C. sordidus.     

Efficacy of cultural control measures against the banana weevil, Cosmopolites sordidus (Germar), in South Africa

The banana weevil, Cosmopolites sordidus, is the most important insect pest of banana and plantain in the world. Cultural control methods were investigated over 2 years in southern KwaZulu‐Natal, South Africa. Harvesting at ground level and dissection of remnants (treatment 1), and covering the base of the mat (entire plant consisting of several meristems) with soil and moving debris to the inter‐row (treatment 2), were compared to a positive control that involved treatment of plants with a registered pesticide (treatment 3), and a negative control that involved harvesting at 150 cm from the collar with no soil or sanitation amendments (treatment 4). Yield, weevil damage and pseudostem girth of plants were measured from August to November annually, while adult beetle densities were assessed over 4 weeks in October/November and April. Nematode samples were taken and analysed in October/November every year. Damage parameters included the coefficient of infestation, the percentage coefficient of infestation (PCI) at two intervals, the summed PCI value, the percentage cross‐sectional damage of the central cylinder and cortex, and the mean cross‐sectional damage percentage. A randomized block design with three replicates was used in the trial. The parameters were similar before the onset of the trial. Fruit yield and plant girth, corrected by nematode densities, were not significantly different in any treatment, nor were the nematodes controlled. Soil cover and recession of remnants was the only effective treatment, significantly reducing the CI, but not the adult density or the other damage parameters. Soil cover showed promise as a cultural control method because it only needs to be applied seasonally and reduced the percentage cross‐sectional damage of the central cylinder, the damage parameter most closely related to yield, by 14%.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Crystal structure of human cystatin D, a cysteine peptidase inhibitor with restricted inhibition profile

Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg(26)-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-A resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel beta-sheet wrapped around a five-turn alpha-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes.     

Proteolytic profile in the larval midgut of Chilo suppressalis Walker (Lepidoptera: Crambidae)

Chilo suppressalis is a key constraint on production of rice. The current research was conducted to study the types of digestive proteases in the larval midgut of C. suppressalis. It was found that activity of total digestive proteases increased from the first to the fifth larval instars, which showed different nutritional requirements. Four types of proteinases and two types of exopeptidase were identified so that their activities from the highest to the lowest activities is trypsin‐like, chymotrypsin‐like and elastase for proteinases, and amino and carboxypeptidases for exopeptidases. Meanwhile, just one type of cysteine protease, cathepsin D, was determined in the fourth and fifth instar larvae. The optimal pH for activity of total protease was found to be pH 9–10 and optimal temperature was observed to be 35–40°C, where there was the highest proteolytic activity. Some specific inhibitors of proteases including PMSF, TLCK, TPCK, DTT, E‐64, cystatin, phenanthroline and EDTA were used to confirm the types of proteases in the midgut of C. suppressalis.