The adult phase of insects' life is primarily for reproduction of young ones that makes continuity of life possible. The influence of reproductive activities like mating and oviposition were investigated in adult males and females variegated grasshopper, Zonocerus variegatus. The adult stage was divided into four phases according to activities performed following days of emergence, namely: early somatic phase, late somatic phase, copulation and oviposition. During each phase, the insects were dissected and the somatic tissues (haemolymph, fat body and femoral muscles) were removed analyzed for both organic and inorganic substances. The mean concentration of organic substances (protein, glucose and lipids) and inorganic substances (Na , K ,ca2 , and Cl-) in both sexes' tissues increased significantly (p < 0.05) from early somatic to late somatic phase. However, there was a significant decrease in concentration of the metabolites in the three tissues during copulation in both sexes which further decreased during oviposition in female adult. In contrast to the female, there was increase in the concentration of the metabolites after copulation in the male adult. Copulation and oviposition are activities that exhaust tissues nutrients in adult Zonocerus variegatus.
The African grasshopper, Zonocerus variegatus (L.) is a tropical insect that belongs to Order Orthoptera and Family Pyrgomorphidae. In Nigeria, it usually occurs on uncultivated land with the nymphs and adult stage sharing the same habitat which extends from rain forest zone to the Guinea Savannah in the north (Youdeowei 1974).
The life cycle of Z. variegatus is built around 6 nymphal stages (Chapman et al. 1977). Nymphs' development is usually completed in 3–5 mo while the adult life takes 15 wk in the laboratory (Muse 2003) which is enough time for egg maturation, oviposition and remating. Nevertheless, the complexity and size of Z. variegatus increases during post embryonic development.
The description and morphometrics of the nymphs of Z.variegatus was reported by Chapman et al. (1977). The antennae increased in length with each instar as well as the number of annuli. Ademolu et al. (2009) observed that the weight, length and width of the hind femur increased as the insect increased in age with the adult life recording a significantly higher size and weight compared to lower instars.
Chemical composition of the tissues of Z. variegatus was examined by Ademolu et al. (2007), who discovered that the concentration of the organic and inorganic substances in the tissues increased during post embryonic development. Previously Modder (1977) had observed that variations existed in the concentrations of metabolites of the somatic tissues of the penultimate instar of Z. variegatus.
Z. variegatus is a polyphagous insect that causes serious damage to both food and cash crops in West Africa (Toye 1982). However, not all food plants contribute to its growth and development (Tamu 1990). For instance, while Manihot esculenta Crantz (cassava leaves) supports the growth, Chromolaena odorata (L.) R. M. King & H. E. Robins and Aspilia africana C. D. Adams do not. Likewise, in a study by Idowu & Sonde (2004) it was found that the survivability and longevity of nymphs and the reproductive performance of adult Zonocerus was enhanced in insects fed on cassava leaves compared to those fed on Acalypha wilkesiana Müll. Arg. and Carica papaya L.
The alimentary canal system of Z. variegatus is divided into 3 parts, i.e., foregut, midgut and hindgut, but major digestion and absorption of nutrients take place at the midgut (Modder 1984). Similarly, the activities of glycosidases, proteinase and lipase increased with age during post-embryonic development (Ademolu & Idowu 2011). In a recent study (Idowu et al. 2009) it was observed that the micro flora present in the gut regions of Z.variegatus were able to produce digestive enzymes that assist the insect in neutralizing the toxic effect of cyanogenic glycosides present in its major food plant.
Mating and oviposition are two energy sapping necessary processes that influence the longevity and development of adult insects including Z. variegatus (Gibbs et al. 2009; Muse 2003). There has not been any report on the effect of reproductive activities on tissue physiology of Z. variegatus. The few existing reports have focused on the effect of reproductive activities on weight and longevity. Thus the thrust of this present study is to examine the influence of reproductive activity on the tissues of both sexes during the adult life.
MATERIALS AND METHODS
Insect Collection
The penultimate (6th) instar stage of Z. variegatus was collected from uncultivated farmland at the University of Agriculture, Abeokuta (UNAAB), Nigeria. They were reared in wire cages (30 × 30 × 45cm) placed at the insectary of the Department of Biological Sciences of UNAAB. They were maintained on fresh leaves of cassava (M. esculenta) until they reached adult stage.
Experimental Procedure
As soon as the insects molted into the adult stage, they were separated into different cages. 20 adult insects were placed into each cage (10 males and 10 females). There were 12 cages and a total of 240 insects (120 males and 120 females). The cages were divided into 4 groups with 3 replicates according to the phases of life during the adult stage or days after emergence as follows: early somatic phase (3–5 d); late somatic phase (10–15 d); copulating phase (23–35 d) and oviposition phase (45–58 d) as described by Idowu & Modder (1998). Each cage has 4 special holes containing sand for oviposition by the females.
At each phase, 10 insects (5 males and 5 females) were removed and dissected for tissue collection. Insect samples for early and late somatic phases were retrieved from their cages at d 3 and d 10, respectively, after emergence. Samples for copulation were collected at d 30 after emergence (they were taken after mating for 6 h), while at d 50 (few hours after oviposition) insect samples for oviposition phase were taken.
Tissue Collection and Preparation
Fat body
The insects were dissected as described by Youdeowei (1974) and the fat body was collected following the method of Modder (1984). The fat body from the body wall was removed with forceps and 5 g of the sub-sample was weighed into a test tube using a sensitive weighing balance (Mettler-PM-11-K) and homogenized in 5 mL of distilled water and the homogenate was kept in the freezer for further analysis.
Hemolymph
The hemolymph was collected by the method described by Ademolu et al (2007). A microneedle was inserted into the mid-ventral axis of the thorax and the haemolymph oozing out was collected into a calibrated syringe, and 100 µL of each haemolymph sample was centrifuged at 1300 rpm. for 15 min to spin down the hemocytes and debris. The supernatant was used for further analysis.
Femoral Muscle
The femoral muscles were collected following method described by Ademolu et.al. (2009). The hind femora was opened with a sharp razor and all the femoral muscles were removed by forceps into the Petri dishes, dried to constant weight at 50 °C in an oven for 12 h. 0.5 g of the sub-samples was vortexed in 0.05 M KCl. The homogenate was centrifuged at 500 rpm (5°C) for 30 min. The supernatant obtained was kept in the freezer for further use.
Chemical Analysis
Organic Substances
The protein content of the tissues was determined by the method of Henry et al. (1997), while the Baunmniger (1994) method was adopted for glucose determination. Lipid assay was done by the method of Grant et al. (1997).
Inorganic Substances
For these analyses, samples were digested with a mixture of per chloric acid and nitric acid (1:2 v/v) and cooled to room temperature. Na+ and K+ were determined by flame photometer while Ca2+ was determined using an atomic absorption spectrophotometer (Model AA.403). The Henry et. al. (1997) method was used to assess the PO42 and Cl- contents of the tissues.
The analyses were done in triplicates and the data were subjected to analysis of variance; and when significance differences occurred, mean separation was done by the Student-Newman-Keul test (SNK).
RESULTS
Hemolymph
The mean concentration of the organic and inorganic substances in the haemolymph of adult females increased significantly (P < 0.05) from early somatic phase to the late somatic phase of the insect. However, there was a decrease in concentration during the copulation and oviposition phases (Table 1). In the Zonocerus adult males similar observations were recorded, i.e., increased concentration during somatic phases and a significance decrease during the mating phase. However, there was increase in concentration of the metabolites during the post copulatory phase in adult males.
Fat Body
The mean concentration of the metabolites in the fat body of adult females increased from the early somatic phase to late somatic phase, but dropped during copulation and oviposition (Table 2). In adult males, no significant difference occurred in the concentration of the fat body metabolites between the somatic phases on one hand and the copulatoory and post copulatory phases on the other hand.
Femoral Muscle
The changes in concentrations of metabolites in the femoral muscles of adult males and adult females followed same patterns as described above for fat body and haemolymph (Table 3). Comparison of means, however, showed that significantly higher lipid concentrations were recorded in the femoral muscles of both adult female and male Zonocerus than in their fat body and haemolymph.
DISCUSSION
Reproductive activities have a significance influence on the physiology of Z. variegatus (Muse 2003); and the present study agrees with this finding. During somatic phases of adult Z. variegatus concentrations of both organic and inorganic substances increased from early to late somatic phases. Idowu & Modder (1998), likewise, had observed increases in the concentration of haemolymph protein during somatic phase. This increase in metabolite concentrations might be due to the buildup of substances necessary for the sexual activity lying ahead of the insect, because the adult stage is mainly devoted to reproductive processes.
The concentrations of metabolites in tissues in both sexes dropped during mating and also during oviposition in the adult female. During mating, the male releases a spermatophore, which contains nutrients, and this contributes to the male's exhaustion. Mating has been described as an energy sapping exercise that affects the weight and fat-body nutrients of adult Melontha melontha (L.) (Leopold 1976). Ofuya et al. (2008), reported that mated males of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) had significantly shorter life spans than unmated males, and, probably, this is due to the energy used by mated male for reproductive activities.
Mating females are assumed to have built up concentrations of metabolite substances preparatory to reproduction, but their non-eating habit and the stress of carrying their partner for hours during copulation might have exhausted or consumed the nutrients gained from the male. Himuro & Fujisak (2010) had earlier reported that mating is costly for both sexes of the seed bug. Togo hemipterus (Scott) (Heteroptera; Lygacidae).
Oviposition similarly reduced the concentration of tissue metabolites of Z. variegatus adult females. During oviposition weight is reduced and less food is eaten (Mccaffery et al. 1978). Muse (2003) observed that Zonocerus females without access to an oviposition substrate (thus could not lay eggs) lived longer than females that oviposite. Mortality was very high after the first oviposition owing to the stress of oviposition activities, which involve the stretching of the abdominal segments (Chapman & Page 1979). The oviposition process, which includes digging the soil is common in Z. variegatus. This digging activity is likely be responsible for the decreases in concentrations of tissue metabolites.
In contrast to the female, the concentrations of tissue metabolites increased during the post-copulatory phase in Z. variegatus adult males. After mating, while females were busy with egg production and preparation of oviposition sites, the adult males resumed normal feeding and thus replenished nutrients lossed during mating.
TABLE 1.
THE CONCENTRATIONS OF METABOLITES IN THE HAEMOLYMPH OF MALE AND FEMALE ADULT Z. VARIEGATUS (MG/DL).
TABLE 2.
THE CONCENTRATIONS OF METABOLITES IN THE FAT BODY OF MALE AND FEMALE ADULT Z. VARIEGATUS (MG/DL).
TABLE 3.
THE CONCENTRATIONS OF METABOLITES IN THE FEMORAL MUSCLES OF MALE AND FEMALE ADULT Z. VARIEGATES (MG/DL).
In conclusion, copulation and oviposition have remarkable impacts not only on body weight but also on the somatic tissues of Z. variegatus.
