The potential of mass rearing of Monoksa dorsiplana (Pteromalidae) a native gregarious ectoparasitoid of Pseudopachymeria spinipes (Bruchidae) in South America

In Chile and Uruguay, the gregarious Pteromalidae (Monoksa dorsiplana) has been discovered emerging from seeds of the persistent pods of Acacia caven attacked by the univoltin bruchid Pseudopachymeria spinipes. We investigated the potential for mass rearing of this gregarious ectoparasitoid on an alternative bruchid host, Callosobruchus maculatus, to use it against the bruchidae of native and cultured species of Leguminosea seeds in South America.The mass rearing of M. dorsiplana was carried out in a population cage where the density of egg-laying females per infested seed was increased from 1:1 on the first day to 5:1 on the last (fifth) day. Under these experimental conditions egg-clutch size per host increased, and at the same time the mortality of eggs laid also increased. The density of egg-laying females influenced the sex ratio which tended towards a balance of sons and daughters, in contrast to the sex ratio of a single egg-laying female per host (1 son to 7 daughters). The mean weight of adults emerging from a parasitized host was negatively correlated with the egg-clutch size, i.e. as egg-clutch size increased, adult weight decreased.All these results show that mass rearing of the gregarious ectoparasitoid M. dorsiplana was possible under laboratory conditions on an alternative bruchid host C. maculatus. As M. dorsiplana is a natural enemy of larval and pupal stages of bruchidae, the next step was to investigate whether the biological control of bruchid C. maculatus was possible in an experimental structure of stored beans.     

Pythium seed decay and stem rot of cowpea (Vigna sinensis [Linn.] Savi) in southern Niǵria

Summary Pythium aphanidermatum (Edson) Fitzpatrick has been associated with seed decay and severe stem rot of cowpea in Nigeria. The symptoms produced on both seedlings and established cowpea plants are described. Fungus-induced losses of 0.94 per cent as seed decay and 11.10 per cent damage to adult cowpea plants have been recorded.     

Biological activity of tree marigold, Tithonia diversifolia, on cowpea seed bruchid, Callosobruchus maculatus (Coleoptera: Bruchidae)

The powder and ethanol extract of Tithonia diversifolia leaves were tested for their efficacy at five different concentrations (0.0%, 0.5%, 1.0%, 1.5% and 2.0%) on bruchid mortality, oviposition and adult emergence of cowpea seed beetle Callosobruchus maculatus at ambient tropical storage conditions of temperature and relative humidity. Results showed that the leaf extract had a higher bioactivity on oviposition, adult emergence and mortality of C. maculatus. The mean number of eggs laid on seeds treated with extract was reduced from 20.7 in the solvent-treated to 4.7 at 2%Tithonia extract concentration while adult emergence reduced from a mean of 92.2 in the solvent-treated seeds to 72.2 at 2.0% extract treatment. Mortality was 100% at higher concentrations of 3%, 4% and 5% within 24 h of extract application but at lower concentrations mortality was 73.3% and 93.3% at 1% and 2%, respectively after 24 h. Fourty-eight hours after application, 100% mortality of adult C. maculatus was obtained at all concentrations. The mean number of eggs laid was reduced from 41.3 in the untreated to 17.3 at 2.0% powder concentration while adult emergence dropped from 98.5% in the control to 74.2% at the highest powder concentration. The powder was effective at higher concentrations and longer exposure time. At 3%, 4% and 5%, 63–75% mortalities occurred within 24 h of application while at 48 h, mortality was 76–98% at 3–5% powder application rates. The results of this study revealed T. diversifolia as a potential candidate for bioinsecticide preparations because of antiovipositional, ovicidal and knockdown properties of its products, which have some volatile components.     

Response of cowpea bruchid,Callosobruchus maculatus (Fab.) (Coleoptera: Chrysomelidae) to cheese wood,Alstonia boonei De Wild stem bark oil extracted with different solvents

Cheese wood, Alstonia boonei De Wild stem bark was extracted with five different solvents namely: methanol, ethanol, acetone, petroleum ether and n-hexane. The extracts were tested for insecticidal activity on cowpea bruchid, Callosobruchus maculatus (Fab.). Adult mortality and adult emergence of the insects were investigated. Oviposition deterrences using dual-choice and multiple-choice tests were also investigated as well as percentage damage and weight loss at temperature of 28?±?2?°C and 75?±?5% relative humidity. Results showed that at the rate of 2% extract per 20?g of cowpea seeds, methanol, petroleum ether and n-hexane extracts caused 100% mortality of adult cowpea bruchid after four?days of post treatment. When the bruchid had choice of oviposition substrate, they laid significantly (p?<?0.05) fewer eggs on cowpea seeds treated with extracts compared to untreated seeds. There was no adult emergence in seeds treated with methanol, ethanol, petroleum ether and n-hexane extracts and 100% reduction in F₁ progeny was recorded compared with untreated that had 81.86% adults emergence and 0% reduction in F₁ progeny. The results obtained from this study revealed that methanol, ethanol, acetone, petroleum ether and n-hexane extracts of A. boonei stem bark were effective in controlling C. maculatus and could serve as an alternative to synthetic insecticides for the protection of stored cowpeas against bruchids.     

Insecticidal potential of cashew (Anacardium occidentale L.) for control of the beetle,Callosobruchus subinnotatus (Pic.) (Bruchidae) on bambarra-groundnut (Voandzeia subterranea L.) Verde

Abstract

The nut and shell of cashew (Anacardium occidentale L.) were evaluated for insecticidal efficacy at concentrations of 2.5, 5.0 and 7.5% per 150 g bambarra groundnut (Voandzeia subterranea L.) Verde seeds against Callosobruchus subinnotatus (Pic.) (Bruchidae). Cashew nut shell was highly toxic to C. subinnotatus and achieved 100% insect mortality within 48 hours at 7.5% and 100% mortality within 72% hours at 2.5 and 5.0% concentrations. Oviposition and progeny development of the insect were severely suppressed while seeds were protected from damage by the bruchid. Seed germination was, however, impaired at all concentrations, suggesting that seed treated with cashew products may be suitable for consumption but not be suitable as planting stock.     

Resistance to the cowpea weevil (Callosobruchus maculatus) larva in pods of cowpea (Vigna unguiculata)

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7% to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%.Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.     

Laboratory and field investigation of a mixture of Metarhizium acridum and Neem seed oil against the Tree Locust Anacridium melanorhodon melanorhodon (Orthoptera: Acrididae)

The in vitro compatibility of Metarhizium acridum strain IMI 330189 with different concentrations of Neem seed oil Azadirachta indica A. Juss. was investigated under laboratory conditions. Water, diesel and ground nut oil dilutions of M. acridum were inoculated into semi-synthetic culture medium with Neem oil and incubated for 10 days at 20, 28, and 34°C. Fungus vegetative growth and conidia production were estimated and compatibility calculated according to the in vitro classification model [T]. Field tests were also conducted during two successive years, where fourth instar nymphs of the Tree Locust, Anacridium melanorhodon melanorhodon, were sprayed with Metarhizium, Metarhizium/Neem mixture, Neem, or Malathion, on a plantation of Acacia senegal. Samples of the treated nymphs were taken 24 h after application, placed in cages and their mortality observed daily for 3 weeks. The compatibility test revealed that at 28°C, all Neem concentrations below 2% were compatible with M. acridum and concentrations of 2 and 2.5% were moderately toxic, while at 20°C, 1.0% Neem was not compatible with the fungus. In the field, Metarhizium+Neem resulted in 74 and 92% mortality during two successive years compared to 64 and 83% for Metarhizium alone. These findings clearly reveal that Neem concentration on the mixture could be increased to just under 1.0% Neem without negative impact on the fungus and that mixing Metarhizium with small quantities of Neem oil accelerates locust mortality and increases efficacy.     

The comparative efficacy of some botanicals for the control of the seed weevil Caryedon serratus Olivier (Col., Bruchidae) in seeds of Acacia seyal Del.

A comparative study on the efficacy of powder and oil extracts from leaves and seed kernels of Azadirachta indica Juss. and Eucalyptus camaldulensis Dehn. against the seed weevil Caryedon serratus Olivier infesting seeds of Acacia seyal Del. was carried out. EO 1, EO 5 and NSKO5% were comparable to BHC 3% in significantly reducing egg laying by C. serratus. With the exception of ELP and NLP, all botanicals significantly reduced egg hatching. Botanicals significantly reduced larval development with the exception of ELP and NLP. EO and NSKO at 3 and 5% were significantly more effective than BHC in reducing larval development by 95–97%. All botanicals had no significant effect on adult emergence which was only affected by BHC. Botanicals caused 4.3–43% mortality of C. serratus adults and the maximum mortality occurred in EO 3% as compared to BHC. It is apparent that oils extracted from E. camaldulensis had a fumigant and knock down effect on adult C. serratus. Extracted oils were more effective than leaf or seed powder. Oils extracted from leaves of E. camaldulensis were volatile and had a knock down effect on adult C. serratus indicating the possibility of using them as alternative fumigants in seed stores.     

Protection of cowpea,Vigna unguiculata L. (Walp.) with Newbouldia laevis (Seem.) extracts against infestation by Callosobruchus maculatus (Fabricius)

Evaluation of protectant ability of Newbouldia laevis (Seem.) extracts against infestation by Callosobruchus maculatus in cowpea, Vigna unguiculata L. (Walp.) was carried out in the laboratory at ambient temperature of 28?±?2?°C and 70?±?5% relative humidity. Extracts from wood ash, leaf, stem and root bark were tested at different concentrations of 0, 1, 2, 3, 4 and 5%. One hundred per cent mortality of adult beetles was achieved at all concentrations within 72?h of treatment with extracts except in wood ash at 1% concentration, but they were significantly different (p?<?0.05) from the controls. All the extracts were still able to cause high beetle mortality after one, two and three months of cowpea storage at high concentrations (4 and 5%) except wood ash, although there was a slight decrease in mortality during the period of storage. All the extracts significantly (p?<?0.05) reduced oviposition and adult emergence of C. maculatus when compared with the controls although the reduction was higher at 5% concentration than others. Adult beetle emergence was completely prevented at higher concentrations (4 and 5%) except in wood ash. Both oviposition and adult emergence increased during the months of storage probably because of the slight reduction in the effectiveness of the extracts. The root bark extract was much more effective in reducing oviposition and adult emergence than others throughout the period of storage. The plant extracts of N. laevis was able to protect the cowpea seeds from damage and prevent weight loss. Cowpea seed damage and weight loss was significantly more (p?<?0.05) in the controls than other for the three-month duration probably because of the more adult emergence. The extracts from N. laevis did not adversely affect the germination of the protected seeds and seed germination ranged from 86.7 to 100%. It has been shown in this study that the extracts of N. laevis were effective against C. maculatus in cowpea although the root bark extract seemed to be the most effective while the wood ash extract was least effective. Their effectiveness, however, slightly decreased during the period of storage. N. laevis could be incorporated into pest management of stored cowpeas since the products are ecologically safe.     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmooLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Why Callosobruchus rhodesianus causes limited damage during storage of cowpea seeds in a tropical humid zone in Togo

The sympatric bruchids Callosobruchus rhodesianus (Pic) and Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) colonize cultures of cowpea, Vigna unguiculata (Walp.) (Papilionaceae), in Togo at the end of the rainy season. Seeds containing larvae of the two bruchid species were introduced into the storage systems. Callosobruchus rhodesianus adults emerged from the seeds at the beginning of storage, but this species disappeared quickly and C. maculatus became dominant. Analysis of the reproduction of females collected at the beginning of storage made it possible to distinguish between four groups of females differing in their reproductive activity. Three groups were sexually active but they differed in their fecundity and the duration of the latency period before the first oviposition phase. The fourth group of females was in reproductive diapause. In interspecific competition, the presence of C. maculatus larvae reduced the survival chances of C. rhodesianus and therefore is disadvantageous for this species. The main factors explaining the rapid decrease of the C. rhodesianus populations under storage conditions were the variability of female reproductive activity with the emergence of insects in reproductive diapause and the significant larval mortality in interspecific competition with C. maculatus.     

Antifungal activity of different plant extracts against Drechslera bicolor causing leaf blight of bell pepper

Aqueous extracts of 48 plants belonging to six different major groups of the plant kingdom, two commercially available botanicals and different fungicides were screened for antifungal activity against Drechslera bicolor causing leaf blight of bell pepper. The test fungi were isolated from bell pepper leaves collected from Udaipur district, Rajasthan, India. Among several botanicals, maximum inhibition of fungal growth was obtained by marigold, lat jeera, lemon grass, mehandi, onion and neem, respectively. Neem oil was superior over Zatropin against the fungus. Vitavax was also found as the best fungicide followed by Quintal and Saaf against the test fungus. The results revealed that these plants could be exploited for ecofriendly management of the diseases caused by the test fungal pathogen and seed biodeterioration during storage.     

The dynamics of laboratory populations ofCallosobruchus chinensis andC. Maculatus (Coleoptera: Bruchidae) in patchy environments

Experiments are described showing the long-term dynamics of two species of bruchid beetles (Callosobruchus chinensis and C. maculatus) in arenas in which the resource of 50 black-eyed beans is divided between 5, 10 or 50 ‘patches’. Both species of adult beetles exhibit clumped distributions between patches. Within a patch there is a tendency for a density dependent reduction in (1) eggs laid per female, (2) the proportion of eggs hatching per bean (C. chinensis only) and (3) larval survival which is strongly overcompensating (particularly in C. maculatus). A discrete generation model is used as a framework to draw these results together and show how the different factors affecting natality and mortality can influence the population dynamics. Finally, the importance of the resource renewal interval in influencing the period of the population cycles is discussed.     

Fusion of a soybean cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically

Abstract 1 The soybean cysteine protease inhibitor soyacystatin N (scN) and Griffonia simplicifolia lectin II (rGSII) have defense functions against the coleopteran cowpea bruchid beetle Callosobruchus maculatus. However, the ability of the insect to activate scN‐insensitive digestive proteases and the relatively low potency of rGSII have hindered their practical application in plant protection. 2 Recent research suggests that defense proteins may achieve increased toxicity and durability when used in combination. Based on the structures of several natural toxin molecules, we hypothesized that covalently linked scN and rGSII could exhibit greater anti‐insect efficacy than the mixture containing individual proteins. 3 To test this hypothesis, a recombinant scN‐rGSII fusion protein that retained both protease inhibitor and lectin functions was constructed. 4 When fed to cowpea bruchid, this new protein showed a synergistic delay in insect development, whereas a mixture of the separate proteins only showed an additive effect. 5 Our results suggest that tethering digestive protease inhibitors to gut epithelium‐interacting lectins could give plant protection superior to strategies based on single genes or mixtures of single gene products.     

Extracts from Tephrosia vogelii for the protection of stored legume seeds against damage by three bruchid species

Aliquots of powdered dried leaves of Tephrosia vogelii (Leguminosae – Papilionoideae) were extracted with hexane, acetone and ethanol. The extracts were tested for their potential to protect stored legume seeds from damage by the bruchids Acanthoscelides obtectus, Callosobruchus maculatus and Callosobruchus chinensis (Coleoptera – Bruchidae). While the acetone and ethanol extracts were ineffective except for a significant but low effect on A. obtectus, the hexane extract had a relatively high efficacy, providing similar control of all three bruchid species. This effect was manifested in slowly‐reduced adult survival within 7 days and in reduced numbers of eggs laid and of F₁ progeny produced, resulting in seed damage averaging 7.1% compared to 99.8% in the untreated controls after one generation. These findings are discussed in the context of previously identified constituents of T. vogelii, the rotenoids rotenone, tephrosin and deguelin.     

Effect of a trypsin inhibitor from Dimorphandra mollis seeds on the development of Callosobruchus maculatus

Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil, are pests that damage stored seeds. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (DMTI-II) isolated from Dimorphandra mollis seeds was tested for anti-insect activity against Callosobruchus maculatus larvae. The inhibitor produced ca. 67% mortality to this bruchid when incorporated into an artificial diet at a level of 1%. The doses necessary to cause 50% mortality (LD₅₀) and to reduce weight by 50% (ED₅₀) for DMTI-II were ca. 0.50% and 0.60%, respectively. The action of DMTI-II on C. maculatus larvae may involve the inhibition of trypsin-like activity of larval midgut extracts, the absence of digestion by midgut preparations or with a mixture of pepsin and papain, and its association with a chitin column and chitinous structure in the midgut of this insect.     

The role of chemical cues in host finding and acceptance by Callosobruchus chinensis

We studied the response of female Callosobruchus chinensis to chemical cues emitted by cowpea seeds at different stages of bruchid infestation (uninfested, egg carrying, L1-, and L4-infested). Olfactory attractiveness was determined in Y-tube olfactometer assays by testing individual seed categories against either clean air or uninfested seeds. Oviposition preferences between uninfested and infested seeds were determined in petri-dish choice-experiments. The olfactometer assays revealed that weevils discriminate between seeds containing different stages of developing bruchids on the basis of olfactory cues. While odors from uninfested and egg-carrying seeds acted as attractants, odors from L1- and L4-infested seeds failed to induce a positive response by the bruchids. When given a choice between uninfested and infested seeds in the olfactometer, weevils preferred uninfested seeds over L1- and L4-infested seeds, but failed to distinguish between uninfested and egg-carrying seeds. In the oviposition experiment as well, bruchids showed a distinct preference for uninfested seeds when offered in combination with L1- and L4-infested seeds. This experiment further showed a reduced acceptance of egg carrying seeds. Our results indicate that C. chinensis females use chemical information during both host searching and host acceptance. Volatiles from uninfested or egg carrying seeds act as attractants, while deterrence increases as development of bruchid immature stages progresses.     

Chemical Composition of the Essential Oil of Nigeria Grown Hoslundia opposita Vahl (Lamiaceae) Dried Leaves and Its Bioactivity against Cowpea Seed Bruchid

Due to several ecological and human hazards of synthetic pesticides in postharvest crop protection, there is the need to search for eco‐friendly alternatives. In this study, chemical composition and insecticidal activities of essential oil (EO) obtained from Hoslundia opposita dried leaves were evaluated against cowpea seed bruchid. Eight constituents, predominated by oxygenated monoterpenes (78.86%), were identified using Gas Chromatography (GC)/MS. The constituents were 1,8‐cineole ( 1 ; 61.15%), followed by α‐terpineol ( 2 ; 16.81%), β‐phellandrene ( 3 ; 13.24%), β‐farnesene (4; 3.55%), α‐pinene ( 5 ; 1.89%), Germacrene D ( 6 ; 1.83%), cis‐sabinene hydrate ( 7 ; 0.90%) and caryophyllene ( 8 ; 0.63%). In fumigation bioassay, at 6 h after exposure (HAE), 0.78 ml EO/l air caused 35.33% mortality which was significantly lower than 60.90% and 63.6% observed at 3.15 and 6.25 ml/l air, respectively. Mortality reached 90.0% at 24 HAE regardless of the applied concentration. Lethal time for 50% of the bruchids (LT₅₀) at concentration of 0.78 ml/l air (6.89 h) was higher than the LT₅₀ at 3.15 and 6.25 ml/l air (4.72 and 4.44 h, respectively). H. opposita EO reduced Callosobruchus maculatus oviposition, while progeny emergence observed in EO‐treated seeds (2.42 – 25.73) was significantly (P < 0.05) lower than 51.56 observed in control. The results confirm H. opposita EO's potentials for control of cowpea bruchids.     

Comparative effects of some botanicals for the control of the seed weevil Caryedon serratus Olivier (Col., Bruchidae)

Abstract:   Comparative efficacy of eco-friendly botanicals extracted from leaves and seed kernel of Azadirachta indica A. Juss (neem) and Eucalyptus camaldulensis Dehn was tested against the noxious weevil Caryedon serratus Olivier. Leaf powder, seed kernel powder and oil extracted from the seeds of A. indica and leaf powder and oil extracted from the leaves of E. camaldulensis and benzene hexachloride (BHC) were tested at 1, 3 and 5% (w/w or v/w) against C. serratus . Eucalyptus leaf oil (ELO) and neem oil (NO) at 3 and 5% were as efficient as BHC and significantly (P=0.0001) reduced egg laying by C. serratus, whereas Eucalyptus leaf powder (ELP) had no significant effect. Neem seed kernel powder (NSKP) at 5%, ELO (3 and 5%) and NO (3 and 5%) significantly (P=0.0001) reduced egg hatching more than BHC at all doses. NO (3 and 5%) and ELO (3 and 5%) significantly (P=0.0001) reduced larval development more than BHC, whereas ELP and Neam Leaf Powder had no significant effect. Adult emergence and mortality were also significantly (P=0.0001) affected by some botanicals tested, especially oils. Oils were more effective as compared with powder. The study indicated that the botanicals tested have a good potential in replacing conventional pesticides because of their high efficacy and environmental safety.     

Synergistic effects of geographical strain,temperature and larval food on insecticide tolerance in Callosobruchus maculatus (F.)

This study investigated the multifactorial interaction of various environmental factors including geographical strain (Brazil, Cameroon and Yemen strains), temperature, dose and larval food (cowpea and mungbean) on the response of Callosobruchus maculatus adult to insecticide. All the main factors, their two‐way interactions and the four‐way interaction had significant effects on C. maculatus response to malathion (an organophosphate insecticide). However, the three‐way interactions were not statistically significant (except strain × food × dose, P = 0.002). The Brazil strain was the most responsive to temperature irrespective of the larval food type. The impact of food type differs from one strain to the other; for instance, the food that imparts higher tolerance in a strain might reduce the tolerance in another. Likewise, the hierarchy of tolerance among the cowpea‐reared strains (Brazil > Cameroon > Yemen) was totally different from that of the mungbean‐reared strains (Cameroon > Yemen > Brazil). The reasons for these differences were discussed in the light of their impact on C. maculatus management. Both the management of C. maculatus and development of resistance could be complex; hence, the states of a variety of environmental factors need to be considered. This is necessary to maximize management success of this bruchid, especially in tropical/subtropical developing countries.