Annual biomass production of two acridids ( Orthoptera : Acrididae ) as alternative food for poultry

Acridids could be an alternative nonconventional protein source for livestock industries like poultry farms. For a high acridid biomass production, selection of acridid species along with their suitable food plants is essential. The present experiment was conducted by rearing of two common Indian acridids, Oxya hyla and Spathosternum prasiniferum, to estimate which one could produce higher annual biomass when fed on three food plants, Dactyloctenium aegyptium, Brachiaria mutica and Cynodon dactylon, to determine the most favorable food plant. Fecundity, fertility, nymphal mortality and sex ratio of both acridid species fed with three different plants were estimated for the annual biomass production in terms of number. Annual biomass production in terms of wet weight, dry weight and energy content were also estimated. Among the two acridid species, O. hyla showed higher values for fecundity, fertility and both wet and dry body weight; and lower values for nymphal mortality in all the three food plant fed sets. Among the three food plants, B. mutica was found to be the most suitable for annual biomass production of both acridids. It was concluded that mass rearing of O. hyla fed on B. mutica could yield a high annual biomass in acridid farms. Additional key words: acridid farm; Brachiaria mutica; fecundity; mass rearing; Oxya hyla.


Introduction
Insects produce a significant biomass in nature and play a major role in animal nutrition, e.g. one attractive and important natural food source for birds are insects.McHargue (1917) reported that the wild bird diet consists almost entirely of insects during the season when they are present.Studier & Sevick (1992) published and survival rate.Nutrient contents of both acridids gave satisfactory results (Anand et al., 2008b).With this information in mind, the multivoltine acridid Oxya hyla (Serville) was chosen for the present study along with Spathosternum prasiniferum (Walker) that had been already used in preliminary studies.Uvarov (1966) observed an increased fecundity of acridids that were fed on plants of Poaceae family.Haldar et al. (1995) also noticed that this plant family was mostly preferred by acridids.On another hand, commonly known plants under Poaceae family are mainly grain crops (rice, wheat, sorghum etc.).Hence it is inconvenient to use these grain crops as food plants for grasshoppers as this may increase the production cost of acridids due to a high expense in sowing and cropping and competition with human food.Although the three species chosen for this experiment belong to the family Poaceae, these are actually abundant weeds which could be easily harvested in grasslands in and around the area of the present study.
The aim of this study was then to determine the effect of three wild plant species of the Poaceae family on biomass production of the selected acridids, in order to find suitable plants and acridid species by which high biomass could be yielded in acridid farms.

Collection and rearing of acridids under laboratory conditions
Two grasshopper species, O. hyla and S. prasiniferum, were collected from nearby agricultural fields and grasslands of Santiniketan (23°39' N, 87°42' E), India, using an insect net of 30 cm diameter.Collected individuals were sent to the Orthoptera section of the Zoological Survey of India, Kolkata, for taxonomic identification.Then the acridid species selected for the present study were reared in the insectaria of the Entomology Research Unit, Dept. of Zoology, Visva-Bharati University, adopting the strategies proposed by Haldar et al. (1999).The collected insects were acclimatized in specially designed cages made of nylon net gauge on wooden frame measuring 75 × 52.5 × 30 cm 3 under laboratory conditions ranging from 30 °C to 35 °C temperature, 70 to 80% relative humidity, 500 ± 25 lux light intensity and 14L/10D photoperiod.Enamel trays measuring 10 × 10 × 4.5 cm 3 filled with fine, washed and sterilized moist-sand were kept inside the cages for that insects are an excellent source of nitrogen, potassium, sodium, iron and magnesium and they fulfill the nutritional requirements for growth and reproduction of birds.Among insects, acridids (short horned grasshoppers under the family Acrididae of the order Orthoptera) which comprises locusts and grasshoppers are known to have a high nutritional value (De-Foliart et al., 1982;Ramos-Elorduy, 1997), high reproductive potential (Haldar et al., 1999;Lomer et al., 2001), rapid life cycle (Ananthakrishnan et al., 1985;Henry, 1985) and higher growth rate (Uvarov, 1966;Scoggan & Brusven, 1972;Muralirangan & Ananthakrishnan, 1977;Ananthakrishnan, 1986).Ramos-Elorduy (1997) reported that protein content of grasshopper varies from 52.1% to 77.1%.McHargue (1917) conducted a proximate analysis on dried Melanoplus spp.(Acrididae) showing 75.3% protein, 7.21% fat, 5.61% ash and high amount of lysine, stating that they might afford a new protein source for poultry and other livestock rations.DeFoliart et al. (1982) and Ramos-Elorduy (1997) estimated that acridids have a high nutritional value and can be used to formulate good quality feed for livestock.Moreover Wang et al. (2007) formulated high protein diets with the acridid Acrida cineria and proved that it is an acceptable feed for broiler without any adverse effect on weight gain, feed intake or gain: feed ratio.
Conventional protein source of poultry feed are mainly fishmeal and grains like maize and soybean which are also natural food for human beings.Nevertheless, resources of these food materials are limited, though their demand is very high.In this scenario these food materials are being overexploited.So, there is a need of alternative protein sources for poultry feed.Acridids may serve as alternative, nonconventional and protein rich, natural feed source for livestock industries like poultry farms, thus overexploitation of the conventional food sources could be controlled.But livestock industries need a constant supply of this alternative food source, which requires establishment of acridid farms that would produce huge biomass of acridids (Ganguly et al., 2010).
Preliminary works on biomass production of Oxya fuscovittata and Spathosternum prasiniferum fed with a single type of food plant were done by Haldar et al. (1999) and Anand et al. (2008a) to utilise this acridid resource as an alternative feed for livestock.Haldar et al. (1999) established that these acridid species are very easy to grow and could produce a huge biomass according to their high reproductive ability Biomass production of acridids insect oviposition.Eggs were collected every third day from the trays of oviposition and transferred to sterilized-sand filled plastic cups at the depth of 2.5-5 cm and incubated at 35 ± 2 o C.After hatching the hatchlings were used for the experimental purpose.

Nymphal mortality, sex ratio and body weight
The following description of the experimental setup is for single acridid species fed with single type of food plant.All the other experiments were carried out with similar setup.Two hundred and fifty newly hatched nymphs of the selected species were collected from insectaria and were kept in five transparent plastic jars measuring 20 × 20 × 35 cm 3 with 50 individuals in each.Then a selected species of food plant were offered in 5 replicates.On achieving the adult stage the number of individuals in each jar was counted to find out the nymphal mortality percentage.On the basis of newly emerged males and females, sex ratio was determined.Males and females of each species were weighed separately with a micro-analytical electro-balance (Sartorius, MC1, Analytic AC120S, Germany).To calculate their dry weight specimens were kept in thermo resistant glass vials and placed in a micro-oven at 45 °C for 72 h.The dried specimens were then reweighed to calculate the dry biomass.

Fecundity and fertility estimation
To find out the effect of food plants on fecundity, single pair of zero-day old adult male and female was kept in transparent experimental jars (25 cm height × 10 cm diameter) for copulation.Three sets of jars with three preferred food plants, i.e.Dactyloctenium aegyptium (L.) Willd., Cynodon dactylon Pers., and Brachiaria mutica (Forsk.)Stapf were maintained for each species.Each set had ten of such experimental jars.The floor of the jar was filled with fresh sterilized sand up to 5.0-7.6 cm from the base.One conical flask (Borosil, 50 mL) filled with water and specific fresh food plant was kept inside the jar and the opening of the jar was covered with nylon net.Sand from the experimental jars was examined every alternate day by sieving with fine mesh to count the egg-pods, if any.Collected eggpods were counted and kept within moist tissue paper in petri dishes for 48 h to get the eggs swollen.Then the frothy material was removed with soft hair-brush-es and forceps.Then the swollen eggs were separated out easily and the number of eggs per egg-pod was counted.Fecundity (total number of eggs laid per female) of each species was calculated by multiplying the number of egg-pods laid per female with the number of eggs per egg-pod.To calculate the fertility (total number of eggs hatched from each female), egg mortality percentage was deducted from fecundity.

Energy content
Deceased adult male and female individuals of both acridid species from the three different food plant fed sets were dried in a hot air oven at 45 °C for 72 h.The dried samples were powdered and mixed with fixed proportion of benzoic acid as burning agent prior to pellet formation.The sample pellets (sample powder + benzoic acid powder) of 1-g weight were charged with Oxygen at 20 kg cm -2 pressure and fired in the Digital Bomb Calorimeter (Model no.RSB-4, Instrumentation India Co., Calcutta, West Bengal) standardized with benzoic acid pellets.The increase in temperature was recorded and the energy content was calculated in terms of calories by following the formula mentioned in the user's manual: where W = the instrument value determined by calibration step, H = specific heat of combustion of benzoic acid, T = temperature increase of water and M = mass of sample.

Biomass production
For the annual biomass estimation only one fertilized female was considered as starting point and then biomass production in subsequent four generations (as both the acridids are tetra-voltine) that could be completed in a year, was estimated by the following procedure.Ten single pairs of a selected acridid were reared under laboratory condition for 1 st generation; with the obtained information the results of the subsequent generations were calculated.For the calculation only viable (on the basis of fertility) nymphs produced from one fertilized female (starting point) in her life span were counted.Then total number of individuals attained adulthood was recorded.In this way biomass of first generation in terms of number of males and females was obtained.Total number of individuals produced from a fertilized female in a year was calculated by adding the number of individuals produced in each of the four subsequent generations for males and females separately.Multiplying the numbers with the mean wet and dry body weight of the individuals of the respective species, biomass production per year in terms of wet weight and dry weight was calculated.Biomass in terms of energy was obtained by multiplying the energy content in kcal g -1 of dry tissue with the total dry biomass (in g) produced per year.

Statistical analysis
Data are presented as means ± SD.Five replicates were carried out for all the variables.For all the selected traits of the chosen acridid species, two-way analysis of variance (ANOVA) were carried out taking food plants and acridid species as factors.For egg-pods laid per female and nymphal mortality, though the factors individually showed significant effect (p < 0.001), their combined interaction did not show any significant variation (p = 0.105).Hence one way ANOVA were carried out for these above mentioned variables at different food plant or acridid species.For annual biomass estimation in terms of wet weight, dry weight and energy content, three way analysis of variance (ANOVA) were carried out taking food plants, acridid species and sex as factors.Duncan's multiple range tests (DMRT) were carried out for each case followed by ANOVA in order to separate the mean values according to significance.All of the analyses were carried out using Microsoft Excel 2000 and S-Plus version 4.0.

Results
The effect of different food plants on the reproductive performance and nymphal mortality of selected acridid species is shown in Table 1.In case of O. hyla, when reproductive variables and nymphal mortality were compared among food plants it was found that B. mutica fed sets produced significantly highest number of egg-pods and these egg-pods had high productivity.The same plant was also found to be the best for S. prasiniferum.On the contrary D. aegyptium fed acridid species showed significantly lower values for all the above mentioned reproductive variables except eggs per pod and fertile eggs per pod of S. prasiniferum, where both traits had insignificant variation between D. aegyptium and C. dactylon fed females.Nymphal mortality (Table 1) between food plants was significantly low in acridid species fed on B. mutica and high in D. aegyptium fed sets.When data were compared within food plants between the acridid species it was noted that O. hyla gave better results for all the mentioned traits except eggs per pod and fertile Within a column a1, b1, c1 and a2, b2, c2 indicate significant differences between mean values within food plants between acridid species and within acridid species between food plants respectively.Two-way ANOVA were done for all variables except egg-pods laid per female and nymphal mortality, where only one way ANOVA were done.DMRT, p < 0.001.Biomass production of acridids eggs per pod of D. aegyptium fed females, where values did not show significant variation.Thus, B. mutica was recorded as the most preferable food plant and O. hyla was the most suitable acridid for better reproductive performance and nymphal mortality.Sex ratio was nearly 2 males: 3 females in O. hyla, whereas in S. prasiniferum males outnumbered females, as the ratio in this species was 4 males: 3 females.
Total number of acridid biomass produced in first generation fed on D. aegyptium is presented in Fig. 1a.In the case of males no significant variation was observed between the acridid species.Female biomass of the first generation was significantly higher in O. hyla than that of S. prasiniferum.In the other three generations (Figs. 1 and 2), both male and female biomass in terms of number followed a similar trend as noticed in female biomass of the first generation.For both acridid species, B. mutica fed sets always gave more number of individuals in all the four generations.
Taking one fertilized female as the starting point, total male and female annual biomass in terms of number produced by each species fed on three selected food plants is shown in Table 2.The trend of annual biomass in terms of numbers of both males and females followed a similar trend as found in each generation (except 1 st ), i.e. between the three different food plants, B. mutica fed acridids gave significantly higher biomass than the remaining two food plants.When the data were compared between the acridids, O. hyla fed on all of the three food plants showed significantly higher values.
The wet weight of acridids is summarized in Table 3.The results showed that among food plants in O. hyla male, B. mutica fed sets gave higher values of wet body weight.A similar trend was observed in S. prasiniferum male.Significantly lower wet weight values of O. hyla and S. prasiniferum were observed in C. dactylon and D. aegyptium fed sets respectively.Between acridids O. hyla gave higher values of wet body weight for all the cases of three food plants.Dry body weight of both acridids followed a similar trend as observed in wet body weight.The energy content of male acridid species was significantly higher in B. mutica fed sets and lower in D. aegyptium fed sets, when data were compared between food plants and within acridid species.On the other hand when the data were compared between acridids, energy content was higher in O. hyla when fed on all the three food plants.
The wet weight of female O. hyla was significantly higher in B. mutica fed sets when data were compared   Within a column a1, b1, c1 and a2, b2, c2 indicate significant differences between mean values within acridid species between food plants and within food plants between acridid species respectively.Two way ANOVA, DMRT, p < 0.001.Biomass production of acridids The effect of different food plants on annual biomass production in terms of wet weight, dry weight and energy content of both acridids fed on the three food plants, is shown in Table 4. Biomass productions in all the above mentioned variables per year in both males and females of the selected acridids were al-

Discussion
High biomass production of acridids in terms of wet weight, dry weight and energy content is a critical factor in establishment of acridid farms in order to mass scale production.Thus the present study was carried out to estimate the ability of acridid biomass production through mass rearing, fed with various food plants.
In India, Iqbal & Aziz (1974) found that acridid species normally feed on some field crops, a few garden plants and weeds.The same authors later reported (Iqbal & Aziz, 1976) the food preference of S. prasiniferum and stated that it selects food plants which are favorable for development and the food preference changes with its age.They also opined that food plants have pronounced effects on development and reproductive potential of S. prasiniferum (Iqbal & Aziz, 1977).Likewise, Fanny et al. (1999) found significant effects on the nymphal period and survival in Oxya nitidula.Ganguly et al. (2010) demonstrated optimum consumption, utilization and development of Oxya fuscovittata when fed on Sorghum halepense.In another species from the genus Oxya, i.e.Oxya japonica Lee & Wong (1978) got significant effects of food plants on the reproductive potential.
Fecundity and fertility are one of the key determinants of mass production.Various authors like Awmack & Leather (2002) and Branson (2003Branson ( , 2006) ) found that host plant quality has an important role in the fecundity of herbivorous insects at both individual and population level.Sanjayan & Murugan (1987) also reported that food plants had pronounced effect on fecundity of Orthoptera.Food quality was a necessary pre-requisite for the development and egg production of acridids (Joern & Behmer, 1997).According to Nezkwu & Akingbohungbe (2002) nymphal development and egg production of acridids are dependent on food plant.The present study also supports the view that food plants exert a great influence on reproductive traits of acridid species.This might be because food plants have significant effects on oocyte development in acridids (Lee & Wong, 1978).Branson (2008) cited that body size of acridids has also an influence on fecundity and fertility, and consequently on their biomass production.Katiyar (1957) reported that among Acrida gigantea and Hieroglyphus assamensis, higher fecundity, fertility and adult emergence were observed in the large species in comparison to the smaller one.Such type of results was also observed in Melanoplus sanguinipes and Melanoplus bivittatus by Smith (1966).Branson (2008) and Anand et al. (2008a) found that fecundity in acridids is positively correlated with body weight and structural size.
In the present study body weight of O. hyla was nearly two times higher than that of S. prasiniferum and fecundity and fertility were consequently higher in O. hyla.When the acridids were fed on B. mutica, large numbers of nymphs were produced.According to Ganguly et al. (2010) nymphal mortality is significantly dependent on food plants.Our results were in concert with this finding because here also food plants and acridid species individually exerted significant effect on nymphal mortality.But their combined interaction did not show significant variation between the two grasshopper species, however, number of viable nymphs was quite high in O. hyla due to their higher fecundity.When starting with a pair of male and female, it was observed that in a year huge numbers of individuals would be produced by B. mutica fed O. hyla due to their high fecundity and fertility.Biomass production (in terms of wet weight) per year was highest in O. hyla (3,415.93kg) when fed on B. mutica.
To calculate yearly biomass production in terms of energy the calorie content of both males and females was estimated.Results showed that in the females of both Biomass production of acridids species, energy was higher than that of males might be due to retention of more energy for reproductive purposes in female.Anand et al. (2008a) also reported that female individual of acridids had higher calorie content than male individuals; they also stated that acridid species of smaller body size had more energy than those of large sized species.A similar trend was observed in our experiment where the smaller species, S. prasiniferum, showed higher energy.However the yearly biomass production in terms of energy was higher in O. hyla (total: males + females = 6,287,527.14kcal) as they had higher body weight and produced large number of acridid biomass.
Results of the present experiment on two common Indian acridid species fed on three different preferable food plants clearly establishes that among the three food plants B. mutica is the most suitable one, and O. hyla could produce a higher annual biomass in terms of wet weight, dry weight and energy content.This information supports the view of establishing acridid farms where O. hyla would be produced in mass scale to provide alternative protein source for poultry and other livestock.However, there exist lacunae in knowledge regarding economic assessment for acridid farm establishment.Hence, it is a must for biologists and economists to come to a single platform for this kind of studies; then only this unconventional protein source could be effectively supplied for livestock feed development.

Figure 1 .
Figure 1.Annual biomass production in terms of numbers of 1 st (a,b,c) and 2 nd (d,e,f) generation of Oxya hyla and Spathosternum prasiniferum fed on three selected food plants (a,d: D. aegyptium; b,e: C. dactylon); c,f: B. mutica).Values are means ± SD.Bars with different letters within a male annual biomass and female annual biomass are significantly different (p < 0.001) using DMRT.

Figure 2 .
Figure 2. Annual biomass production in terms of numbers of 3 rd (a,b,c) and 4 th (d,e,f) generation of Oxya hyla and Spathosternum prasiniferum fed on three selected food plants (a,d: D. aegyptium; b,e: C. dactylon); c,f: B. mutica).Values are means ± SD.Bars with different letters within a male annual biomass and female annual biomass are significantly different (p < 0.001) using DMRT.

Table 1 .
Reproductive potential and nymphal mortality of acridids, fed on three preferred food plants

Table 2 .
Total number of individuals produced annually taking a single pair of male and female as starting point

Table 3 .
Wet weight, dry weight and energy content of Oxya hyla and Spathosternum prasiniferum

Table 4 .
Annual biomass production (mean ± SE) of of both acridids males and females in terms of weight and energy