Cowpea, Vigna unguiculata (L.) Walp., is a drought-tolerant and warm weather leguminous crop that is well adapted to the drier regions of the tropics (Singh, 2005). The grains of this legume are widely consumed and serve as an essential source of plant protein to millions of people in sub-Saharan Africa (SSA) and other parts of the world (Bolarinwa et al., 2022; Ige et al., 2011; Nwagboso et al., 2024). The grains are severely damaged during storage by a bruchid, the cowpea weevil, Callosobruchus maculatus Fab. (Coleoptera: Chrysomelidae) (Hajam and Kumar, 2022; Devi and Devi, 2014). Infestation starts on the field when females get into damaged or shattered pods and lay their eggs on the seeds. Under favourable environmental conditions in storage, these eggs hatch and the weevil population builds up rapidly leading to both qualitative and quantitative grain losses (Singh et al., 1985; Kpoviessi et al., 2019; Torres et al., 2016). Weevil damage is manifested as reductions in seed weight and germination ability, reduced nutritional value, and adverse effects on the appearance of cowpeas, rendering them unfit for consumption and commercialization (Torres et al., 2016). In general, losses due to cowpea weevil infestation can be as high as 100 percent within six months of storage of untreated cowpea (Mkenda et al., 2014).

To effectively mitigate losses due to this pest, the use of hermetic technology, such as the Purdue Improved Crop Storage (PICS) bags, has proved to be an environmentally-friendly strategy (da Silva et al., 2018). Thermal disinfection has also been reported to be effective at managing eggs, larvae, pupae, and adults of this insect although the different stages require different temperatures and different times of exposure (Loganathan et al., 2011). Other environmentally benign strategies include the use of botanicals (Eugenia aromatica (Baill.), Cymbopogon citratus (lemon grass), Citrus sinensis (orange peel) and Azadirachta indica (neem) (Ofuya et al., 2010; Ojebode et al., 2016; Kusuma et al., 2014), Spinosad sprays (Sanon et al., 2010), vegetable oils (Khan, 2011), and microbial pesticide formulations containing Bacillus thuringiensis (Malaikozhundan and Vinodhini, 2018). These management approaches, however, are not widely adopted by resource-poor farmers and others in the cowpea value chain because of limited access to these protection agents and the high cost of some of these management options. For botanical extracts, the labour associated with their preparations often limit their adoption in SSA, while the price of those industrially prepared and marketed ones are prohibitive.

Hence. most farmers, grain merchants and seed producers rely on synthetic insecticides, such as carbamates (propoxur), organochlorines (lindane), organophosphates (e.g., acephate, diazinon, dichlorvos, dursban, malathion, pirimiphos-methyl), synthetic pyrethroids (permethrin), phosphine and phostoxin, methyl bromide, and iodofenphos, to mitigate losses due to the cowpea weevil (Ekoja et al., 2021; Hajam and Kumar, 2022). Insecticide misuse to control bruchids can be the main cause of harmful pesticide residues often found in marketed cowpea grains (Olufade et al., 2014; Olutona and Aderemi, 2019). Residues of these insecticides in the grains, and the development of insect resistance to the active ingredients in their formulations are major concerns. There is therefore a need to increase the quality of stored cowpea by exploring more effective, economic, and environmentally friendly cowpea weevil management alternatives. The development of varieties with resistance to infestation and damage by the cowpea weevil is the most viable cost-effective method of overcoming this damaging storage pest and increasing access to the grains of this nutritious legume (Singh et al., 1985; Kpoviessi et al., 2019; Nyarko et al., 2022). However, to date there is no known conventionally bred cowpea variety with an adequate level of protection from infestation and damage by this weevil. Only a few varieties with modest resistance under low insect pressure have been identified, and this is usually influenced by physical seed attributes such as surface area, smoothness, and curvature (Adam and Baidoo, 2008; Ajayi and Lale, 2001; Musa and Adeboye, 2017), as well as chemical traits such as production of trypsin, arcelin, and p-aminophenylalanine (Amusa et al., 2014; Badii et al., 2013; Onigbinde and Oigiangbe, 1996; Tengey et al., 2023).

In the absence of adequate sources of resistance in conventional breeding programs, biotechnological approaches are needed to introduce effective and durable protection from cowpea weevil infestation and damage. Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by α-glucosidases. Several insects, especially bruchids that feed on starchy seeds during larval stages, depend on their α-amylases for survival. The α-amylase inhibitor 1 (αAI-1) protein produced in the common bean (Phaseolus vulgaris) binds irreversibly to the enzyme active site and inhibits the activity of porcine, human, and insect α-amylases (Altabella and Chrispeels, 1990) and is thus toxic to bruchids in adzuki beans (Ishimoto et al., 1996), peas (Schroeder et al., 1995; Shade et al., 1994), chickpeas (Sarmah et al., 2004), and cowpeas (Solleti et al., 2008; Lüthi et al., 2013). Cowpea line IT86D-1010 was genetically modified via Agrobacterium-mediated transformation to create transgenic event CSI-32, which expresses the kidney bean αAI-1 protein exclusively in the seed as a means of controlling post-harvest damage due to the bruchid weevil.

The current study evaluated the phenotypic characteristics and post-harvest resistance to weevil damage of CSI-32 cowpea in comparison with its non-transformed parental line, IT86D-1010, and three released cowpea cultivars, including SAMPEA 7, SAMPEA 20-T and IT13K-1070-2. The purpose of the field phase was to assess whether there were any adverse effects on agronomic performance or plant phenotype arising from the genetic modification resulting in event CSI-32, while the storage study on harvested grain was intended to evaluate the efficacy of the introduced weevil resistance trait.

Genetic variability among cowpea genotypes is crucial for the efficiency of cowpea improvements (Gerrano et al., 2015). These genetic differences account for observed variations in traits like plant height, flowering time, seeds per pod, productive branches, and seed yield (Shimelis and Shiringani, 2010). Therefore, the agronomic and phenotypic differences seen in the studied genotypes are expected. Similar variations in phenotypic traits among genetically diverse cowpea varieties are well documented (Animasaun et al., 2015; Agyeman et al., 2015; Belay et al., 2017; Nkoana et al., 2019).

The transgenic event, CSI-32, and its near-isogenic conventional comparator, IT86D-1010 exhibited normal growth and development when phenotypic parameters, including plant vigor were measured. The only difference detected between line CSI-32 and the control IT86D-1010 was the resistance to bruchid damage, which was the goal for the introduced trait. The findings suggest that the introduced αAI-1 gene and production of high levels of αAI-1 protein in the seed of transgenic event CSI-32 did not give rise to unintended phenotypic effects. Similar reports of a lack of significant difference in phenotypic characters between the transgenic Cry1Ab-expressing event, SAMPEA 20-T, and control cowpea entries were reported by Addae et al. (2020).

Damage to cowpea by the pod-sucking bug (PSB) complex (C. tomentosicollis, A. curvipes, R dentipes, N. viridula, T. custator and A. armigera) did not differ among the entries. To date, cowpea breeding programmes have not succeeded in developing varieties that are stably resistant to multiple damaging pests of the crop, including PSBs (Togola et al., 2017). This is because several minor genes (polygenic resistance) regulate the control of multiple insect pests’ attacks. Until recently, the introgression of these minor genes into new varieties was considered complex. In recent times however, advances in biotechnology makes it possible to introgress these genes into improved cowpea varieties (Sharma et al., 2002; Kogan, 1994). This notwithstanding, none of the cowpea entries used in this work were resistant to the PSB pest complex. Damage by these sucking pests probably accounted for the differences in the 100-seed weight at Nyankpala in particular.

Apart from SAMPEA 20-T, which expresses the Cry1Ab insecticidal protein conferring resistance to damage by Maruca pod borer (Addae et al., 2020), all the genotypes, including CSI-32, were susceptible to damage by this pest. The expression of αAI-1 protein in CSI-32 seed would not be expected to confer protection against pod borers and the in-field pest observations were consistent with the activity spectrum of αAI-1.

Cowpea yields are impacted by environmental variables (rainfall, temperature), genotypic potential, and pests and diseases (Cerıtoglu and Erman, 2020; Jackai and Daoust, 1986; Ibrahim et al., 2021; Karungi et al., 2000). In this study, a uniform spray regime was applied to all entries to control arthropod pests, and their exposure to elements of the weather were the same. Hence, grain yield was determined largely by the yield potential of the individual genotypes assessed. Insect damage did not significantly affect yield. Although genotype was a significant factor for yield (p<0.001), yields were very similar among four of the five entries, with only IT13K-1070-2 exhibiting higher yield at both locations.

Except landraces (Silva et al., 2021), many studies that have evaluated commercially cultivated improved cowpea varieties for weevil resistance, have been unsuccessful in identifying durable sources of resistance to this voracious pest (Badii et al., 2013; Deshpande et al., 2011; Kpoviessi et al., 2019; Amusa et al., 2018). Similarly, none of the commercial cowpea entries tested in this work were resistant to C. maculatus. The genotype, SAMPEA 7, which is reported to be susceptible to weevils (Azeez and Pitan, 2013; Ojumoola and Adesiyun, 2014; Ofuya and Credland, 1995) and IT13K-1070-2, which is reported to be moderately resistant to this pest (Tengey et al., 2023), were both found to be highly susceptible to weevils when they were subjected to an infestation pressure of 50 sexually matured insects per 200-g seed sample. Oviposition was similar across all the entries tested, including CSI-32, indicating that αAI-1 did not act as a deterrent to oviposition; however, it did act to completely suppress larval development and adult emergence during the 4-month duration of the study.

The median development period (MDP), adult emergence, and severity of grain damage are important determinants of ranking cowpea genotypes as being susceptible to cowpea weevils. Prolonged development time, low adult emergence, and low grain damage are key characteristics of resistant genotypes and vice versa (Amusa et al., 2018; Azeez and Pitan, 2013; Silva et al., 2021). Additionally, Badii et al. (2013) and Amusa et al. (2018) reported a difference in MDP of ca. 3 and ca 6 days, respectively, between susceptible and resistant cowpea genotypes. Here, we report ca. 0.5 days difference between the highest and lowest MDP, which is unlikely to be biologically meaningful. The genotypes, IT86D-1010, SAMPEA-7, IT13K-1070-2 and SAMPEA 20-T, were categorized as being highly susceptible based on mean Dobie’s Susceptibility Index (DSI) values >20 for each entry. Further, the survival and emergence of adults as well as the overall high levels of seed damage recorded for these genotypes confirmed their susceptibility.

This is the first study to examine both in-field agronomic performance and post-harvest storability of a transgenic cowpea expressing αAI-1 to control the cowpea weevil. The findings of the current study and earlier ones clearly demonstrate that developing cowpea varieties that express the αAI-1 protein presents a sustainable and cost-effective approach to mitigating damage by this pest. The technology to control the weevil is incorporated in the seed itself and protection is not reliant on the application of synthetic insecticides that have known adverse health and environmental consequences (Kolawole et al., 2014; Kpoviessi et al., 2019). Further, this technology could complement the use of hermetic sealed bags, especially for those who can afford the bags in Africa (Hajam and Kumar, 2022; Silva et al., 2018).

Genetic modification approaches have proven highly effective at separately controlling key field pests, such as M. vitrata, and as shown here, the post-harvest storage pest, C. maculatus. As discussed in Barrero et al. (2021), future work could include the development of novel cowpea varieties combining these two important insect resistance traits for the benefit of smallholder farmers and consumers in West Africa.