Abstract: Background and Objective: Alphitobius diaperinus (Coleoptera: Tenebrionidae) is the major pest in chicken farms. The control of pests using synthetic insecticides is not recommended. Besides its expensive, synthetic insecticides are proved harmful to poultry health, farmworkers and polluting the environment. To explore the sources of bioinsecticides that are cheap and eco-friendly, this study was conducted to evaluate the insecticidal effect of weeds extracts (Chromolaena odorata, Ageratum conyzoides and Tithonia diversifolia) against post-embryonic survival of A. diaperinus. Materials and Methods: The experimental study using a completely randomized design of 2 factors, i.e., 3 types of weeds extract and 6 levels of concentration. The insecticidal effect was observed based on the post-embryonic survival. The post-embryonic survival of larvae was evaluated for 4 weeks by measuring the number of Larvae Released (LR) from the eggs, as well as the number of Adults Emergence (AE). Data were analyzed using Variance Analysis (ANOVA) and continued with the least significant different tests (α<0.5). Results: The statistical analysis showed that the differences in extract concentration contributed significant influence (α<0.05) on the total number of larvae released and adult emergence. The lowest LR and AE occurred at the 40% concentration level, i.e., on the extract of T. diversifolia, while the highest LR and AE were found in the treatment of C. odorata extract. Conclusion: In this study, all types of extracts have an insecticidal effect on the post-embryonic survival of A. diaperinus but the strongest effect was found in the extract of T. diversifolia in 40% concentration. At last, these findings inform people that the weeds extract, especially T. diversifolia is possible to be developed as bioinsecticides for A. diaperinus.
INTRODUCTION
Most chicken farming businesses in Indonesia are small businesses managed by farmers in rural areas. The warm and humid environment in which the chicken has reared favours the development of several organisms, including the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae). Alphitobius diaperinus is one of the most common insect pests in poultry farms, where it reproduces within the littered floor, feeding on poultry manure, spilt feed and other organic material. It is the most significant pest in broiler houses worldwide1. This insect causes serious economic losses if not adequately controlled, because it can rapidly spread in poultry houses and it may affect birds, breeding facilities and farmworkers, Mature larvae create pupation chambers in cage wall cracks, causing structural damage. A. diaperinus is also difficult for poultry to digest which can cause intestinal lesions2.
Larvae and adults are potential transmitters of several viruses, fungi and bacterial diseases (such as Newcastle disease, Escherichia spp., Fowlpox, Salmonella spp., Aspergillus spp. and Campylobacter spp.), which can lead to poultry weight loss and even death3-7. The presence of these insects disrupts the activity of the chicken, they often scratch the litter and feed on it, so reduction of balanced feed intake. The consumption of adult insects may injury the chicken digestive tract due to their hard elytrons. Adult A. diaperinus will be releasing a secretion as a defence mechanism against predators when disturbed. This secretion is a quinone compound, which is a toxic and carcinogenic substance that may cause liver lesions8.
The use of synthetic insecticides (such as alpha-cypermethrin, spinosad and methyl-pirimiphos), is the most common method to control A. diaperinus, applied by spraying the floor and walls before the replacement of the litter for the next breeding cycle, to avoid direct contact with chicken9,10. However, the use of synthetic insecticides in addition to being expensive for small breeders is also proven to have a noticeable negative impact, often not specific and also affect non-target organisms. Negative impacts that can happen include causing pest resistance, killing non-target organisms, increasing chemical residues in the soil and agricultural products, as well as health problems for humans and livestock11. The loss of field efficacy of synthetic insecticides and resistance of A. diaperinus populations have been reported12-14 and has led to a great interest in the development of eco-friendly insecticides. Therefore, exploration and developing a cheap and environmentally safe source of botanical insecticides (bioinsecticides) is important and indispensable.
Plants are an abundant source of secondary metabolites such as alkaloids, flavonoids, steroids, glycosides, terpenoids, tannins, saponins, quinine and phenols15 and have the best potential as pharmaceutical drugs and biopesticides16. The sources of bioinsecticides that have not been widely disclosed are plants that are not of economic value but are abundantly available, such as post-harvest waste and weed crops. Weeds are a group of wild plants that are considered the main crop disruptors of agriculture, for example, Ageratum conyzoides, Tithonia diversifolia and Chromolaena odorata from the family Asteraceae17. In Indonesia, these weeds are found in corn18 and soybeans19. Weeds can compete with cultivated plants in obtaining sunlight, nutrients and growing space to the detriment of the main crop. Often weeds are superior in competition because weeds have a very high growing speed, the ability to adapt to the environment and can breed vegetatively and generatively20. As with most plants, weeds contain secondary metabolites that serve as self-protection from the attack of predators21. The metabolites compound can be toxic to natural enemies, repellent, antifeedant effect or are allelopathic which can inhibit the lives of other organisms around it. The fact that weeds have the potential to be a source of bioinsecticides is expected to shift its status from a plant with no economic value to a useful plant22.
Thus this study aims to analyze the potential of 3 weed plants (C. odorata, A. conyzoides and T. diversifolia) as reproductive inhibitors of poultry pest A. diaperinus. For small-scale farmers, bioinsecticide source plants that are easy to find around, easy to grow and environmentally friendly will be very useful information economically. The cost of obtaining and processing abundant weed crops near farmland is guaranteed to be cheaper than the cost of purchasing synthetic pesticides.
MATERIALS AND METHODS
Study area: This study was experimental design under laboratory conditions, in the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Semarang from January-April, 2021.
Extracts preparation: The leaves of A. conyzoides, T. diversifolia and C. odorata were collected from Temu Kencana herbal medicinal garden, Magelang. A respective sample of weeds dried and powdered than were emulsified in with ethanol solvents 96% in ratio 1:5 for 3×24 hrs. After 72 hrs the extracted liquid was then filtered using a glass funnel given a filter paper. The filtrate was put in a vaporizing flask, then evaporated with a rotary evaporator at 45°C. The evaporation is continued by using a water bath at 50°C until all solvents were lost and produced a concentrated extract. The concentrated extract was assumed to be a 100% concentration.
Phytochemical screening: Qualitative phytochemical analysis of methanol extracts of A. conyzoides, T. diversifolia and C. odorata was conducted following the standard procedures. The analysis was performed through phytochemical screening of alkaloids, flavonoids, terpenoids, phenolics, steroids, saponins and tannins on extracts.
Insect preparation: Adults of A. diaperinus were collected from commercial poultry houses at Gunungpati Semarang Indonesia. Approximately 100 adults (males and females) were reared in the laboratory and maintained in a plastic container with a perforated cover, in the dark incubators at 27±2°C and 70±5% RH. They were fed with poultry food. Polystyrene sheet was placed in the containers, which served as a site for the larvae to pupate. The F1 progeny were used for treatment and adults with good performance were selected for the bioassays.
Evaluation of the post embryonic survival: The survival of F1 progeny from adult A. diaperinus exposed to weed extract would be observed based on the number of larvae that emergence as well as the number of larvae that successfully became adults, during 4 weeks of maintenance. A plastic cup diameter of 5 cm with 7 cm height was used as a rearing container. The experimental study using a completely randomized design of 2 factors. The 1st factor was the concentration of the extract (0, 10, 20, 30, 40, 50) and the 2nd factor was the source of the extract (C. odorata, A. conyzoides, T. diversifolia). Each treatment was repeated 5 times. Each experimental unit using 3 pairs of adult A. diaperinus. Exposure to the extract was done by dripping 150 μL per 3 pairs into the body of each insect. All of the treatments were rearing with poultry food for 4 weeks at 27±2°C and 70±5% RH. The data taken in this observation was the cumulative number of larvae released, as well as the number of larvae that emergence successfully into an adult.
Statistical analysis: The number of larvae released and the number of adult emergences was analyzed using Variance Analysis (ANOVA). If the results of the analysis showed a significant effect (α<0.05), they would be followed by a different LSD test.
RESULTS AND DISCUSSION
Phytochemical screening of weeds: The result of the phytochemical screening (Table 1) reveals that alkaloid, phenolic and tannins were positive in all ethanolic extracts of C. odorata, A. conyzoides and T. diversifolia. Terpenoids were detected in the ethanolic extract of A. conyzoides and T. diversifolia, while flavonoids were detected only in the ethanolic extract of T. diversifolia as well as steroids were detected only in the ethanolic extract of C. odorata. These phytochemicals may be responsible for their insecticidal properties. The presence of terpenoids revealed that the plants can act mainly as antifeedant and growth disruptor and possesses considerable toxicity toward insects. Thus, the most variation of active compounds found was in T. diversifolia extract.
Generally, the efficacy of bioinsecticides of plant origin that have been widely studied leads more to the efficacy of certain secondary pure metabolite isolates, for example, essential oils. However, it turns out that the bioactivity of extracts with various phytochemical compounds contained in them can be complementary or synergistic. Synergistic effects can occur when the combination of bioactive substances in the extract provides a greater effect than the effects of each component23. The phytochemical activity of extracts consisting of a combination of various metabolite compounds can contribute to the overall biological effects. Synergistic active compounds will increase the effectiveness of insecticides24.
Metabolite compounds in plants that have insecticidal activity are alkaloids, flavonoids, phenols, steroids and terpenoids25. Alkaloids have a carbon skeleton derived from isoprenoids and those that have insect repellent activity are aconitum alkaloids and steroidal alkaloids26. Alkaloids, in addition to causing acute toxicity, cause disruption of biological membranes, internal damage to organs and metabolism, redox imbalances, disorders in the developmental process, reproductive disorders or causes inhibition of food intake in insects27,28.
Terpenoids are aromatic compounds, give off a distinctive odour on each plant and can be used as insect repellents29. Previous studies have proven that the active compounds of terpenoid groups are toxic to insect pests. Although its mechanism of action does not directly kill the targeted insects, it contributes importantly to limiting the spread of the population. Sublethal effects include reduced fertility, shortening life, repellent effect, antifeedant, chemosterilant, growth retardant, oviposition inhibitors and attractant30,31.
| Table 1: | Phytochemical screening of weeds | ||
|
|
Weeds | ||
| Parameters |
C. odorata |
A. conyzoides |
T. diversifolia |
| Alkaloids |
+ |
+ |
+ |
| Steroids |
+ |
- |
- |
| Terpenoids |
- |
+ |
+ |
| Flavonoids |
- |
- |
++ |
| Phenolic |
+ |
+ |
+ |
| Saponins |
- |
- |
- |
| Tannins |
+ |
+ |
+ |
| +: Detected, ++: More detected and −: Undetectable | |||
| Table 2: | Total number of larvae released and adult emergence | |||
|
Extract concentration (%) |
C. odorata |
A. conyzoides |
T. diversifolia |
Average |
|
Total number of larvae released |
||||
| 0 | 69±22.057 | 69±22.057 | 69±22.057 | 69±22.057a |
| 10 | 69±14.533 | 63±7.301 | 54±16.263 | 62±12.699ab |
| 20 | 53±11.000 | 55±18.823 | 49±14.832 | 52±14.855bc |
| 30 | 50±20.401 | 56±13.255 | 40±14.536 | 49±16.064cd |
| 40 | 53±19.766 | 43±8.933 | 22±6.671 | 39±11.790de |
| 50 | 47±9.555 | 36±13.183 | 19±4.764 | 34±9.167e |
| Average | 57±16.218a | 54±13.925a | 42±13.187b | |
|
Total number of adult emergence |
||||
| 0 | 67±21.078 | 67±21.078 | 67±21.078 | 67±21.078a |
| 10 | 66±14.433 | 59±9.524 | 48±15.754 | 58±13.237b |
| 20 | 51±10.780 | 51±20.732 | 45±12.798 | 49±14.770c |
| 30 | 47±17.649 | 53±12.689 | 36±14.536 | 45±14.958c |
| 40 | 49±18.398 | 37±9.680 | 17±6.066 | 34±11.381d |
| 50 | 42±8.136 | 29±11.323 | 12±2.550 | 28±7.336d |
| Average | 54±15.079a | 49±14.171a | 37±12.130b | |
| Different letter in the same printed column shows a significant difference at 5% significance level and based on LSD post-hoc test | ||||
Therefore, it is necessary to examine the effect of the phytochemical compounds of 3 types of weeds on the survival of A. diaperinus larvae.
Evaluation of the post embryonic survival: The postembryonic survival of A. diaperinus was measured based on the number of larvae released as well as the number of larvae that successfully metamorphosed into adults (Table 2).
The statistical analysis showed that the differences in extract concentration contributed significant influence (α<0.05) on the total number of Larvae Released (LR) and Adult Emergence (AE). The lowest LR and AE occurred at the 40% concentration level, i.e., on the extract of T. diversifolia, while the highest LR and AE were found in the treatment of C. odorata extract. Within 4 weeks, the average number of larvae and the number of adults found in the treatment of A. conyzoides extracts didn’t significant from the treatment of C. odorata (α>0.05) but both significant to the number of larvae and adults in the T. diversifolia treatment. The higher the concentration of the extract is, the less the number of larvae will be released. Apparently, the higher the concentration of extracts that are in direct contact with the body of insects is, the greater the chance of the extract being absorbed into the body and disrupting the reproductive tract.
Yasmin et al.32 state that the direct contact, the active compound of the extract can penetrate the insect body through its spiracle organs. The entry of the active compound of neem leaves into the body of A. diaperinus disrupts the oviposition tract and inhibits the process of laying eggs (oviposition deterrent).
Ingrid et al.1 also found that the effects of A. conyzoides extract have affected the histopathology of the reproductive tract of locusts Zonocerus variegatus. On the contrary, Green et al.33 found something different because in their research T. diversifolia extract did not affect the egg-laying ability of C. maculatus (p>0.05). Belmain et al.34 argue that the efficacy of each extract may vary greatly, influenced by plant genetics and growing environmental differences. The dominant components contained in the extract, the species of test insects, the concentration as well as the way the active compounds of the extract entering the body will also affect its efficacy. In this study, a decrease in the number of larvae and the number of adult ticks shows that the three weed extracts of C. odorata, A. conyzoides and T. diversifolia can be used to suppress A. diaperinus population. Tithonia diversifolia extract provides the best results compared to the other 2 weed extracts.
CONCLUSION
All types of extracts have an insecticidal effect on the post-embryonic survival of A. diaperinus but the strongest effect was found in the extract of T. diversifolia in 40% concentration. At last, these findings inform people that the weeds extract, especially T. diversifolia is possible to be developed as bioinsecticides for A. diaperinus.
SIGNIFICANCE STATEMENT
This study discovers the possible synergistic effect of bioactive substances in the weeds extract and provides a greater insecticidal effect than the effects of each component. At least, T. diversifolia expressed the strongest effect because it contains 5 types of secondary metabolites, more than the other 2 weeds. This study will help the researcher to uncover the botanical insecticides that are cheap and eco-friendly that many researchers were not able to explore.
ACKNOWLEDGMENT
The author would like to thank Universitas Negeri Semarang for funding this research, through the 2021 UNNES DIPA fund with Number: 242.26.4/UN37/PPK.3.1/2021.