1 Introduction

Pulses are significant cash crops that support both household food security and economic stability, in the farming community. According to Shukla and Mishra [1], India is the world's biggest producer and consumer of pulses, making up roughly 25% of worldwide production, 27% of consumption, and 34% of food use. The world’s largest producer of mung beans, India accounts for 54% of global production and 65% of acreage. To raise public awareness of the nutritional advantages of pulses in sustainable food production, the Food and Agriculture Organisation (FAO) of the United Nations declared 2016 to be the international year of pulses. One-tenth of the total production and sixteen percent of the area accounted for by mung beans alone. The Indian domestic market for mung beans is expected to reach 2.75 million tonnes by 2030, out of all the pulses (IIPR [2]). According to Baraki et al. [3], around 7.3 million hectares are planted with mung beans globally, with an average production of 721 kg/ha. India and Myanmar together account for 30% of the world's total production of 5.3 million tonnes. Notable producers include China, Indonesia, Thailand, Kenya, and Tanzania (Nair et al. [4]). Insect pests are a major impediment to pulse production, resulting in significant losses during field and storage operations. About thirty percent of India's pulse losses and about $815 million in monetary losses are caused by insect pests (Prakash et al. [5]).

According to Mendki et al. [6] and Tripathy [7], the pulse beetle or bruchid is a pest that is particularly harmful to pulse crops that are stored. When compared to other insects, bruchids are tiny (1–6 mm). Among bruchids, 30 species are known to be severe storage pests on various pulse crops over the world (Mishra et al. [8]). The majority of the bruchids infesting stored pulses are thought to belong to the genus Callosobruchus, which has 16 species that have been indentified. The bruchid beetles, which are found in the tropics and sub-tropics, are mostly found in two species: C. maculatus and C. chinensis (War et al. [9]; CABI [10]). The pulse beetle favors mung beans as one of its hosts. Bruchid infestations in field settings have little impact since the bruchids deposit their eggs on the green pod's surface. However, seeds can suffer significant damage from infection while stored (Tripathi [11]). Seed germination capacity is also decreased, in addition to causing qualitative and quantitative losses. It is observed that up to 60 per cent of weight loss of the stored seed occurs due to pulse beetle (Golnaz et al. [12]). It is very important to understand the bruchid species that severely infesting storage pulses. To determine the identification characters of both the species that live together, the study on morphometric characters of pulse beetle is of utmost important. The reproductive interference between C. maculatus and C. chinensis, focuses mainly on its role in competitive exclusion (Kishi et al. [13]). The present study was carried out to determine morphological biometrics of C. maculatus and C. chinensis and their interspecifics C. maculatus (female) × C. chinensis (male) and C. chinensis (female) × C. maculatus (male) in mung bean.

2 Materials and methods

2.1 Sample collection

C. maculatus and C. chinensis were mass reared on mung bean under laboratory conditions at 27 ± 1 °C temperature with a 65 ± 5% (RH) in the insect growth chamber (IGC) at Banda University of Agriculture and Technology, Banda during 2024. The pure culture of C. maculatus and C. chinensis were obtained from Indian Council of Agricultural Research (ICAR)—Indian Institute of Pulses Research (IIPR), Kanpur. After adult emergence from the infested seeds, male and female adults of both sexes were kept in a polypropylene storage box (250 ml) for mating and allowed for egg laying on freshly harvested seeds of mung bean. Mung bean seeds with freshly laid eggs of C. maculatus and C. chinensis were introduced in 1.5 ml tubes to develop virgin adults for further experimentation. The healthy 50 g seed was kept in plastic tubes (10 × 5 cm) and five pairs (five males and five females) of the newly emerged pulse beetle were released. They were allowed to remain in the grains for mating and oviposition and the beetles were removed from the container five days after the release when they laid the eggs.

The virgin adult male and female were mated interspecifically to develop the offsprings of interspecifics. The one virgin female of C. maculatus and the one virgin male of C. chinensis were kept in a plastic tube with healthy mungbean seeds for mating and egg laying. Similarly, the one virgin female of C. chinensis and the one virgin male of C. maculatus were kept with healthy mungbean seeds in order to lay the eggs. The experiments were carried out with three sets and each set consisted of 10 replications. The observations of egg laying were noticed till the death of insects. Further, the eggs laid on the seeds were kept at 27 ± 1 °C temperature with a 65 ± 5% (RH) in the insect growth chamber (IGC) to develop the adults. The adult emergence was observed up to three months.

2.2 Morphometric observations

The morphometry observations were recorded on various life stages of C. maculatus and C. chinensis i.e. eggs stage, larval instars (first, second, third, and fourth), pupal stage and adult stage (female and male) with the help of ocular micrometers and vernier calliper on three sets of ten replications for each stage. The egg, larval, pupal and adult (female and male) morphometry of both species were recorded at different intervals. In the case of pupa, the observation was recorded at the stage when wing and locomotion appendage were initiated after the fully grown larval stage. The fully mature adult was recorded after emergence when wing and locomotion appendages were completely developed.

To develop interspecifics, males of C. maculatus were caged with females of C. chinensis and males of C. chinensis was caged with females of C. maculatus and observation was recorded for the development of egg and further growth and other developmental stages.

The data were analysed by using OPSTAT Software to determine analysis of variance (ANOVA) and other parameters of statistical significance. The investigation was conducted in compliance with the protocols recommended by Banda University of Agriculture and Technology, Banda.

3 Results

Mung bean was observed to be an appropriate host for C. maculatus and C. chinensis rearing in the laboratory conditions throughout the year. Each developmental stage of both species was observed and the results are presented here as under-discussed with the support of previous findings.

3.1 Egg

Adult females were oviposited single fertilized egg on the external surface of the bean. The eggs, when freshly laid, were translucent, smooth and shining and are dome-shaped structures with flat oval bases which later became yellowish white and very prominent against the contrasting background of the grain surface (Fig. 1). Individually egg was oval or spindle shaped, clear, shiny and firmly glued to the bean surface and had a yellow colour which became opaque when hatched. Eggs were 0.66 ± 0.03 mm in length and 0.36 ± 0.02 mm in width of C. maculatus (Table 1).

Fig. 1
figure 1

a Eggs of C. maculatus & a–i Single egg of C. maculatus. b Eggs of C. chinensis & b–i Single egg of C. chinensis. c Eggs of Intersex (C. maculatus ♀ × C. chinensis ♂) & c–i Single egg of intersex

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Table 1 Morphometric observations and developmental duration of different growth stages of C. maculatus and C. chinensis
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3.2 Larva

Larvae were yellowish-white in colour with reduced legs and a brown head (Fig. 2). They were found mostly inside the seed. The first instar larvae were 0.68 ± 0.12 mm in length and 0.36 ± 0.07 mm in width and second instar larvae were 1.09 ± 0.08 mm in length and 0.61 ± 0.03 mm in width and third instar larvae were 2.46 ± 0.15 mm in length and 1.32 ± 0.10 mm in width and the final fourth instar larvae were 3.58 ± 0.28 mm in length and 2.07 ± 0.14 mm in width of C. maculatus. The first instar larvae were 0.63 ± 0.12 mm in length and 0.34 ± 0.03 mm in width and second instar larvae were 0.97 ± 0.07 mm in length and 0.56 ± 0.05 mm in width and third instar larvae were 1.20 ± 0.10 mm in length and 0.75 ± 0.07 mm in width and the final fourth instar larvae were 2.55 ± 0.18 mm in length and 1.40 ± 0.10 mm in width of C. chinensis.

Fig. 2
figure 2

Larval and Pupal instars of C. maculatus. a First larval instar. b Second larval instar. c Third larval instar. d Fourth larval instar. e Early pupal instar. f Pupal-Adult intermediate

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3.3 Pupa

Larvae burrowed to a position just underneath the seed coat before pupation. Although the seed coat of the mung bean was still intact, a round 1–2 mm window was apparent at the location where the beetle was pupating. The pupae were dark brown in color (Fig. 3). The length and width of pupae were 4.30 ± 0.29 mm and 2.37 ± 0.18 mm of C. maculatus and 2.71 ± 0.18 mm length and 1.48 ± 0.08 mm width of C. chinensis.

Fig. 3
figure 3

Larval and Pupal instars of C. chinensis. a First larval instar. b Second larval instar. c Third larval instar. d Fourth larval instar. e Early pupal instar. f Pupal-Adult intermediate

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3.4 Adult

Male and female pulse beetles (sedentary morph) were easily distinguished from one another by their general appearance (Fig. 4). Body length of C. maculatus female and male adult was 4.28 ± 0.16 mm length and 2.20 ± 0.09 mm width; 3.40 ± 0.17 mm in length and 2.01 ± 0.13 mm in width, respectively. C. chinensis female and male adult were 2.96 ± 0.17 mm length and 1.60 ± 0.14 mm width; 2.26 ± 0.15 mm length and 1.30 ± 0.11 mm width, respectively, in both species.

Fig. 4
figure 4

Adults of. a C. maculatus Male. b C. maculatus Female. c C. chinensis Male. d C. chinensis Female

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The pigmentation on the plate covering the end of the abdomen was the most distinctive feature. The plate was larger and had a black hue on both sides in the female. The plate was smaller and had no stripes in the male. In most cases, male beetles are brown in color and females are black, however in some cases, both sexes are brown. Two pairs of wings; the first pair became the elytra sheathing, which covers only a portion of the abdomen and is darker in color in females than in males (Fig. 5). The elytra protected the membranous, longer hindwings, which were longer than the forewings. The adult male and female C. maculatus elytra measured 1.92 ± 0.06 mm in length and 0.94 ± 0.06 mm in breadth, and 2.21 ± 0.07 mm in length and 0.99 ± 0.06 mm in width, respectively. Elytra of C. chinensis male and female adults were 1.75 ± 0.03 mm length and 0.83 ± 0.03 mm width; 1.90 ± 0.04 mm length and 0.89 ± 0.04 mm width, respectively (Table 2).

Fig. 5
figure 5

Elytra of a C. maculatus Female. b C. maculatus Male. c C. chinensis Female. d C. chinensis Male

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Table 2 Morphometric observations of distinct morphological parameters of C. maculatus and C. chinensis
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The antennae were pectinate in males while in females, the antennae were serrate (Fig. 6). The female beetle's antennae were shorter than those of the male. Antennae of C. maculatus male beetle were 1.70 ± 0.08 mm in length and the female beetle was 2.09 ± 0.08 mm length, respectively. Antennae of C. chinensis male and Female beetle were 1.35 ± 0.05 mm in length and 1.24 ± 0.03 mm in length, respectively.

Fig. 6
figure 6

Antennae of a C. maculatus Female. b C. maculatus Male. c C. chinensis Female. d C. chinensis Male

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Pygidium of C. maculatus male and female adult were 0.96 ± 0.06 mm length and 0.77 ± 0.05 mm width; 1.06 ± 0.05 mm length and 0.87 ± 0.05 mm width, respectively (Fig. 7). Pygidium of C. chinensis male and female beetle were 0.81 ± 0.07 mm length and 0.73 ± 0.04 mm width; 0.98 ± 0.06 mm length and 0.89 ± 0.05 mm width, respectively (Table 2).

Fig. 7
figure 7

Pygidium of a C. maculatus Female. b C. maculatus Female (dissected part of pygidium to abdomen). c C. maculatus Male. d C. chinensis Female. e C. chinensis Female (dissected part of pygidium to abdomen). f C. chinensis Male

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3.5 Interspecifics’ of C. maculatus and C. chinensis

It was observed in the present investigation that females of C. maculatus and males of C. chinensis did occasionally mate, however, males of C. maculatus and females of C. chinensis rarely did so. The freshly laid eggs were very minute, translucent smooth and dull coloured with flat oval bases, firmly glued to the bean surface and all the eggs could not hatch to develop the larvae. It may be concluded that the eggs of intersexes were infertile. The eggs of cross between C. maculatus female and C. chinensis male were 0.60 ± 0.03 mm in length and 0.34 ± 0.01 mm in width.

4 Discussion

Each developmental stage of both the species was observed and discussed with the support of previous findings. In C. maculatus eggs were 0.47 ± 0.08 mm in length and 0.12 ± 0.21 mm in width, the first, second, third and fourth instar larvae length were 0.60 ± 0.03 mm, 1.19 ± 0.05 mm, 2.43 ± 0.15 mm, 3.64 ± 0.18 mm and width were 0.22 ± 0.03 mm, 0.77 ± 0.02 mm, 1.22 ± 0.08 mm, 2.00 ± 0.11 mm), respectively, the pupa of C. maculatus was 4.57 ± 0.07 mm in length and 2.60 ± 0.06 mm in width and the adult female and male were 3.70 ± 0.12 mm in length and 2.17 ± 0.05 mm in width and 3.21 ± 0.06 mm in length and 1.91 ± 0.05 mm in width, respectively, have been reported by (Devi and Devi [14]). In C. chinensis the average egg size was 0.30 ± 0.12 mm in length and 0.18 ± 0.07 mm in width, the first, second, third and fourth instar larvae length were 0.83 ± 0.12 mm, 0.95 ± 0.10 mm, 1.07 ± 0.05 mm, 1.49 ± 0.15 mm and width were 0.37 ± 0.06 mm, 0.49 ± 0.04 mm, 0.62 ± 0.05 mm, 0.92 ± 0.07 mm), respectively, the pupa of C. chinensis was 1.56 ± 0.21 mm in length and 0.83 ± 0.09 mm in width and the adult female and male were 2.16 ± 0.26 mm in length and 1.21 ± 0.12 mm in width and 2.02 ± 0.27 mm in length and 1.17 ± 0.10 mm in width, respectively, are reported by (Jui et al. [15]). The species specific variations in the present and past investigation might be due to different bruchid culturing conditions.

The antennae of C. maculatus male beetle were 1.81 ± 0.07 mm in length and the female beetle was 1.49 ± 0.07 mm in length and elytra of male and female beetle were 2.12 ± 0.02 mm in length and 0.99 ± 0.04 mm in width and female were 2.20 ± 0.14 mm in length and 1.10 ± 0.08 mm in width reported by (Singh and Swamy [16]). The antennae of male beetle were 2.02 ± 0.14 mm length and the female beetle was 1.71 ± 0.12 mm length of C. maculatus and antennae of C. chinensis male beetle were of 1.89 ± 0.11 mm length and the female beetle was 1.54 ± 0.08 mm length (Colgoni and Vamosi [17]).

The length and width of the elytra of male C. maculatus were found to be 2.11 ± 0.02 mm and 0.98 ± 0.04 mm, respectively while, in the case of female it had a slightly larger elytra with 2.18 ± 0.14 mm length and 1.08 ± 0.08 mm width (Premkumari et al. [18]). The length and width of the elytra of males C. chinensis were found to be 1.84 ± 0.13 mm and 0.83 ± 0.059 mm, respectively, whereas for females, 1.93 ± 0.11 mm and 0.86 ± 0.074 mm (Colgoni and Vamosi [17]), similarly, (Kafom et al. [19]) also reported that the maximum length of elytra was found to be 1.73 ± 0.14 mm in Senegal. Elytral length and width of male were 1.74 ± 0.08 mm and 0.82 ± 0.02 mm, respectively, (Premkumari et al. [18]), length and width of elytra were found to be 2.21 ± 0.01 mm and 1.21 ± 0.01 mm, respectively for males and 2.65 ± 0.02 mm and 1.38 ± 0.01 mm, respectively for females on mung bean (Kashyap et al. [20]). The variations observed in the present investigation and in the findings of previous workers might be due to different experimental conditions.

The pygidium of male beetle was 0.94 ± 0.08 mm length and 0.77 ± 0.07 width and the female beetle was 1.19 ± 0.10 mm length and 0.93 ± 0.09 mm width Singh and Swamy [16]. Similarly, Premkumari et al. [18]) reported that the average pygidium length and width of C. maculatus for female C. maculatus were determined to be 1.18 ± 0.10 mm and 0.92 ± 0.09 mm, respectively and 0.95 ± 0.08 mm and 0.78 ± 0.07 mm, respectively in male and the average pygidium length and width for female C. chinensis were found to be 0.92 ± 0.09 mm and 0.84 ± 0.08 mm, respectively in males and 1.06 ± 0.11 mm and 0.98 ± 0.10 mm in females.

The females C. maculatus and males C. chinensis did occasionally mate, however, male C. maculatus and female C. chinensis rarely did so the interspecifics between females C. maculatus and males of C. chinensis finding is also supported by the findings of Kyogoku and Sota [21]; Yamane and Miyatake [22] who reported that females C. maculatus and males C. chinensis did occasionally mate. The male intromittent organ of C. chinensis is narrower than that of C. maculatus, and thinner intromittent organs from C. chinensis may more easily enter the female reproductive tract of C. maculatus (Kyogoku [23]).

The delineation of morphological variations in between C. maculatus and C. chinensis will give a clue in sound identification of the pulse beetle and through which the species specific magnitude of damage can be assessed more easily.