Histopathology of the Midgut (Longitudinal section) of Aedes aegypti larvae after exposure to kaffir lime leaf extract from Bali, Indonesia

 

Hebert Adrianto, Etha Rambung, Hanna Tabita Hasianna Silitonga

School of Medicine, Universitas Ciputra, Citra Land CBD Boulevard, Kel. Made RT 04 RW 01,

 Kecamatan Sambikerep Surabaya 60219, East Java, Indonesia.

*Corresponding Author E-mail: hebert.rubay@ciputra.ac.id

 

ABSTRACT:

Various countries have reported the resistance of mosquitoes to synthetic insecticides and larvicides. Scientists take advantage of secondary metabolites from plants and develop them into natural larvicides. Kaffir lime leaves (Citrus hystrix) as a cooking spice is of concern in this study. The main aim of this study was to identify differences in midgut histopathological changes in Ae. aegypti larvae after exposure to C. hystrix leaf extract. This research is experimental research in the laboratory. There are five treatments with three replications. Then, 20 third-instar Ae. aegypti larvae were placed into each plastic container and recorded for 24 hours. Histopathological slices of the larval midgut were produced and stained with hematoxylin and eosin (HE). A light microscope was used to identify it. Data on changes in midgut larvae were analyzed using SPSS. Mortality of the larvae was shown in C. hystrix extract groups at doses of 1,500 and 3,500 ppm. Midgut changes occur in the length of the midgut lumen, the length of the epithelium, and the loss of the epithelial cell nucleus. The cell nuclei were not visible in the extract group at a dose of 3,500 ppm. The statistical test showed that there are differences in midgut histopathological changes in Ae. aegypti larvae after exposure to C. hystrix leaf extract. We need an in-depth study of the potency of the extract C. hystrix as an alternative larvicide against Ae. aegypti for the future.

 

KEYWORDS: Citrus hystrix, Midgut, Histopathology, Longitudinal, Aedes aegypti.

 

 


INTRODUCTION: 

Dengue fever is an arboviral infection and mosquito-borne disease of major public health importance that is particularly prevalent in tropical and subtropical climates.1,2 There are four DENV serotypes (DENV-1 to DENV-4) that cause dengue fever. This disease is transmitted through the intermediary of the genus Aedes mosquito.3–5 The Aedes mosquito is also capable of transmitting diseases other than dengue, such as zika, yellow fever, and chikungunya.6–8 Dengue symptoms can range from an asymptomatic infection to a severe infection with multi-organ failure.3,4 Dengue infection most often occurs in the rainy season. Dengue infection in Indonesia often causes outbreaks, illness, mortality, and economic losses.9,10

 

There is still a lack of effective treatment or vaccines to treat dengue, causing mosquito control to remain one of the priorities of the Ministry of Health.11–13 Mosquito control tactics are based on the life stages of the mosquito (eggs, larvae, pupae, and adults). Mosquito management methods include spraying chemical insecticides at adult mosquitoes or destroying mosquito larvae before they mature with synthetic larvicide (temephos). Repellents are used to prevent mosquito bites.14,15 Temephos, a synthetic larvicide, has been established as a method for the Ae. aegypti eradication program in Indonesia since 1980.16 For a long time, the use of synthetic larvicide has resulted in a variety of issues, including environmental contamination, larvicide resistance, and hazardous dangers to humans. Many countries have reported the ineffectiveness of synthetic larvicides against Aedes aegypti larvae.17–20 The development of resistance against larvicides in Ae. aegypti led to operational failures in control programs.18

 

Scientists are currently starting to develop alternative larvicides from plants. Plant extracts are being used as alternative larvicides in studies because they include a number of particular phytochemicals that kill mosquito larvae without affecting other creatures or the environment.21,22

 

Kaffir lime (Citrus hystrix) leaves are a plant that is popularly used by Indonesian people as a cooking spice.23,24 Kaffir lime leaves contain chemical compounds such as essential oils, flavonoids, saponins, and steroids. They work as poison on the larvae.24 Citronellal, Citronellol, 3-Carene, linalool, and citronellol are the primary chemical components found in the essential oil of kaffir lime leaves.25,26 Because this plant has been used economically, its safety is no longer in doubt. Our previous studies have reported the potential of kaffir lime leaves (C. hystrix) from Indonesia as an Ae. aegypti larvicide. However, our study is still limited to bioassays on larval mortality.24 The mosquito gut consists of a tube that is divided into three separate structures, namely the foregut, midgut, and hindgut. Midgut functions in the digestion of food and the absorption of nutrients.27 The midgut becomes the target organ of the larvicide and causes the larvae to die.28–30 To date, how C. hystrix leaves overcomes the midgut is poorly understood, despite being an important subject matter. In this study, we reveal the effect of kaffir lime (C. hystrix) leaf extract from Bali on changes in the midgut of Ae. aegypti larvae.

 

This study aims to identify differences in midgut histopathological changes in Ae. aegypti larvae after exposure to C. hystrix leaf extract.

 

MATERIALS AND METHODS:

Materials:

Preparation of Methanol Leaf Extract of C. hystrix:

C. hystrix leaves were obtained from Banyupoh village, Gerokgak District, Buleleng Regency, Bali Province, Indonesia. Fresh leaves are then dried for 1 month and ground into powder. C. hystrix leaf methanol extract was prepared by maceration of C. hystrix dry leaf powder in a glass jar. The maceration solvent is methanol. After two days, the maserate was filtered. The resulting filtrate was evaporated using a rotary evaporator to obtain a black, viscous extract. The eggs of Ae. aegypti was obtained from the Laboratory of Entomology, Surabaya Ministry of Health Health Polytechnic, Surabaya, East Java province, Indonesia. The eggs hatch into larvae, which are cared for until they reach the 3rd instar larvae.

 

Larvicidal test:

The experiments were carried out at room temperature in the Research Laboratory at Universitas Ciputra, Surabaya. C. hystrix leaf extracts were tested against Ae. aegypti mosquito 3rd instar larvae. There are five treatments with three replications. Four treatments of C. hystrix extracts were produced with concentrations of 80, 400, 1,500, and 3,500ppm. Each replicate was placed in a 500ml plastic container with 200ml of C. hystrix leaf extract. The negative control treatment group contained 200ml of aquadest and 5drops of Tween 20. The number of dead and live larvae in each container was then counted, and the percentage mortality was calculated after 24hours of treatment.

 

Histopathology Study Evaluation:

Six larvae from each treatment were preserved in 5 mL of 10% formaldehyde and sent to the Biosains Institute at Universitas Brawijaya in Malang, East Java Province, Indonesia. They were placed in a 10% Formalin solution at room temperature for 24hours while still alive. They were properly washed with PBS (Phosphate Buffer Saline) after this interval to remove any residue. Larvae were dehydrated by immersing them in a series of escalating ethanol concentrations for 15 minutes, commencing with 50%, 70%, 80%, 90%, and 95%, then 100% ethanol twice for 30 minutes, then overnight. Tissue-Tek TEC 5 Sakura was used to embed the larvae in paraffin (Sakura) (embedding and Cryo Module). The embedding cast was prepared in stainless steel molds (10mm; Sakura), with one larva positioned lengthwise in each block. To guarantee that the molds were hot enough for the process, they were held at 62°C for at least 30 minutes. A manual microtome (Accu-Cut SRM Sakura) with disposable blades was used to cut longitudinal sections of larval midgut (3m) (MX35 Ultra Thermo Scientific). These slides were stored in a Hotplate (Sakura) at 40°C for at least 24hours. Lastly, the slices were stained with Mayer's hematoxylin and eosin Y for histological examination. The histology slides were viewed and photographed using an Olympus microscope (BX53). The scale bar was calculated using ImageJ software version 1.53t. The length of the epithelium cell, the length of the lumen, and the presence of the nucleus of the epithelium cell all related to the observed midgut damages.

 

Statistical Analysis:

Statistical analysis was done with SPSS version 26. Differences in the length of the epithelium will be analyzed using the Kruskall-Wallis test followed by the Mann-Whitney test to determine which dose gives a significant difference to the length of the epithelium because the data is not normally distributed and does not have a homogeneous variance. Differences in the length of the lumen will be analyzed using the one-way ANOVA test, which is then followed by a post hoc test, namely the LSD test, to find out which dose gives a significant difference to the length of the lumen. Presence of epithelial cell nuclei is presented qualitatively.

Ethical approval:

This study was approved by the Ethical Committee of Universitas Ciputra's School of Medicine in Surabaya, Indonesia, as detailed in Ethical Clearance No. 036/EC/KEPK- FKUC/ II/ 2023.

 

RESULT:

According to the findings of this study, there were no dead larvae in the control group and 100% mortality in the C. hystrix extract group (doses of 3,500ppm). Extracts with concentrations of 80 and 400ppm have not caused mortality. The concentration of the extract at 1,500ppm causes 50% larval mortality. The concentration of the extract at 3,500ppm causes 100% mortality. The effects of the control group and the treatment group on the histopathological changes of the midgut are presented in Table 1.

 

Table 1. Histopathological change in the midgut of Ae. aegypti larvae

Group (Larva condition)

The mean epithelial cell length (΅m) + SD

The mean midgut lumen length (΅m) + SD

80 ppm (lived larva)

1.20 + 0.03

5.79 + 0.02

400 ppm (lived larva)

1.23 + 0.03

 6.09 + 0.11

1,500 ppm (lived larva)

1.52 + 0.06

5.38 + 0.63

1,500 ppm (dead larva)

1.38 + 0.03

6.81 + 0.25

3,500 ppm (dead larva)

0.84 + 0.09

5.56 + 0.68

Akuades (lived larva)

1.25 + 0.09

5.71 + 0.73

Kruskall Wallis (* = Significantly different at the real level 5%)

 

Differences in the length of the epithelium will be analyzed using the Kruskall-Wallis test followed by the Mann Whitney test to determine which dose gives a significant difference to the length of the epithelium because the data is not normally distributed and does not have a homogeneous variance. The results of the Kruskall Wallis statistical test showed that the five treatment doses had significantly different epithelial lengths (p = 0.005 <0.05). The results of the Mann Whitney test found that the dose that produced the lowest epithelial length was a dose of 3500ppm of dead larvae. In the second order, there are groups of doses of 80ppm, 400ppm, and distilled water on live larvae. In the third order, there is a dose that produces the lowest epithelial length, namely 1500ppm of 50% dead larvae. In fourth place, namely the dose of 1500ppm, 50% live larvae.

 

The results of the one-way ANOVA statistical test showed that the five treatment doses had significantly different lumen lengths (p = 0.003 <0.05). The extract dose that provided the lowest midgut lumen length was the dose group of 1500ppm (50% live larvae), 3500ppm (dead larvae), distilled water (live larvae), 80ppm (live larvae), and 400ppm (live larvae). The five doses provided the same lumen length and were not significantly different. Whereas the 1500ppm dose group (50% dead larvae) had the lowest lumen length after the previous 5 doses and the temephos dose group had the highest lumen length compared to the other dose groups.

 

 

Figure 1. Longitudinal section part of 3rd instars larvae of Ae. aegypti larvae midgut exposed to C. hystrix extract for 24 hours, stained with H&E (magnification, Χ400). lm= lumen, N: nucleus of epithelial cells, EC: epithelial cells. A= 80 ppm (lived larva), B = 400 ppm (lived larva), C = 1.500 ppm (dead larva), D= 1.500 ppm (lived larva).

 

In the larval midguts that were exposed to high doses of extract, namely 3500ppm, no cell nuclei of epithelial cells (N) were visible. This appearance was different from the treatment group at a dose of 40 ppm to 1500 ppm, where nuclei were still found in the midgut cells. Likewise, the negative control group also found intact and large cell nuclei (Figure 2)

 

 

Figure 2: Longitudinal section part of 3rd instars larvae of Ae. aegypti larvae midgut exposed to C. hystrix extract (3,500ppm) (upper figure) and control group (lower figure)

 

DISCUSSION:

The ability of C. hystrix causes larval mortality:

Our study proves that C. hystrix leaf extract has a mortality effect on Ae. aegypti larvae. Previous studies also proved the same thing that C. hystrix and Citrus genus leaf extracts had a mortality effect on mosquito larvae. Sarma et al. discovered that essential oil from the bark of C. grandis had a higher LC50 value of 61.71 ppm than essential oil from the leaves (LC50 value of 126.45ppm at 72hours).31 Kaffir lime peel essential oil from Kuncen Banguntapan Bantul Yogyakarta at a dose of 103ppm were reported to be effective in killing larvae by 96%.32 Various types of citrus have been compared and tested for their activity as larvicides, such as the leaves of C. hystrix, C. amblycarpa, and C. maxima. Kaffir lime leaf extract (C. hystrix) has the highest toxicity compared to both lime leaf extract (C. amblycarpa) and grapefruit leaf (C. maxima).24 Plants in the rutaceae family group, such as Chloroxylon swietenia, Citrus aurantifolia, Citrus limon, Citrus reticulata, Citrus hystrix, Citrus sinensis, Clausena excavate, Feronia limonia, Ruta graveolens, Swinglea glutinosa,  Toddalia asiatica, Zanthoxylum armatum, Z. articulatum, Z. avicennae, Z. piperitum, and Z. monophyllum have toxic properties against mosquitoes.33

 

Changes in larval midgut after exposure to the extract:

Rohmah et al. studied the effect of Ae. aegypti fruit extract on the midgut of Ae. aegypti larvae. Columnar cell vacuolization, epithelial nuclei crossing the midgut lumen, microvilli disruption, and basement membrane damage were the most common types of injury detected in the larval midgut.34 Similar results were also found by Liu et al., who reported that the ar-turmerone mechanism is a stomach toxin, with the active site perhaps being muscle and digestive tissue. Ar-mode turmerone's of action is unrelated to AChE.35 Essential oil of kaffir lime peel obtained from the city of Yogyakarta was tested on Ae. aegypti larvae for changes in abdominal color morphology to black. In addition, observation of histological preparations of larvae indicates epithelial cell damage in the midgut.32 The midgut of a healthy larva is composed of epithelial cells, single-layered, cylindrical in shape, attached to a basement membrane, with a globular nucleus situated in the center. The apical surface of the epithelial cells is covered with microvilli. The cytoplasm is pink, the nucleus is stained purple with hematoxylin-eosin staining, and there is a peritropic matrix.32 The key activities of the mosquito larva's midgut are digestion, generation of digestive enzymes, the synthesis and secretion of enzymes and hormones, ion transport, absorption, and osmoregulation processes.33,36 In addition, it also has enzymes such as glutathione S-transferases (GSTs) which play a role in metabolic detoxification processes.37 Midgut cell damage is directly related to dysregulation of digestive enzymes and detoxification. Damage to midgut cells between the treatment group and the control group proves that there is stomach toxic activity from plant extracts.33,36 Damage to the midgut will disrupt the physiological function of the insect.

 

Saponins have larvicide potential and function as stomach poison in Ae. aegypti larvae by reducing the surface tension of the mucosa membrane of the digestive tract, making it more easily injured. Saponins enter the larvae body through the digestive tract. The midgut of larvae was damaged mostly as a result of different functions occurring in this location, such as digestion, nutrition absorption, ion transport, and osmoregulation.34 Plant-derived essential oils have also been found to have a vital role in mosquito control and to have larvicidal properties.38,39 Previous findings reported that essential oils from P. corcovadensis, P. marginatum and P. arborerum caused histological changes in the midgut of S. frugiperda larvae. Essential oils have lipophilic properties that can penetrate the exoskeleton cuticle of insects to affect organs including the midgut and fat body.36 Essential oils such as d-limonene have lipophilic properties, so they can penetrate the insect cuticle. The mechanism of trans-anethole and essential oil toxicity has been reported to cause changes in thoracic pigmentation, partial lysis of midgut epithelial cells, abnormal thoracic cell shape, changes in thoracic shape, and midgut damage. The majority of larvae exposed to d-limonene did not live long. Those who died had morphological abnormalities in their thorax coloring, head and thorax forms, and damage to the midgut, anal papillae, and respiratory siphon. Furthermore, lysis of cephalothorax cells, cuticle color change, and changes in the form of the head, midgut, and respiratory trumpets were seen in Ae. aegypti and Ae. albopictus pupae at the time of death.8 The lipophilic monoterpene content of essential oils can enter the integument, cell wall, and cell membrane and damage the structure of the cell membrane, which can result in cell lysis. This could be due to its function, loss of ions, reduction of the membrane potential, breakdown of the proton pump, or depletion of the ATP pool.37

 

Environmental safety is considered to be of paramount importance. Insecticides do not need to cause high mortality in target organisms to be acceptable, but they must be environmentally friendly.40 This potential is owned by C. hystrix, which is a popular plant in society as a cooking spice. The limitation of this study is that it still uses crude extracts, and the observations are limited to midgut histopathological observations. In the future, molecular research, bioinformatics, molecular docking, and proteomics will be needed to sharpen the study of the extract's activity as a stomach poison.

 

CONCLUSION:

There are differences in midgut histopathological changes in Ae. aegypti larvae after exposure to C. hystrix leaf extract. We need an in-depth study of the potency of the extract C. hystrix as an alternative larvicide against Ae. aegypti, by identifying secondary metabolites, conducting studies at the biomolecular and bioinformatics levels, and then testing them in field trials.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

Funding for this study was supported by the Research and Publication Institute at Universitas Ciputra, Surabaya (004/UC-LPPM/DIP/PDUPT/VII/2022). The authors wish to thank all the personnel, such as Michael Adi Wijaya, Lidya Anin, Eva Maria Anigomang, Intan Murni Arifah, Gede Tegar Witnandika Suara, Krisdiyanti Ellyfas, Pupimadita Tizar, and Yuliyanto Adi Perdana, for their kind assistance

 

REFERENCES:

1.      Bimal MK, Kaur L, Kaur M. Assessment of knowledge and practices of people regarding dengue fever. Int J Nurs Educ Res. 2016; 4(2): 174-178. doi: 10.5958/2454-2660.2016.00035.1.

2.      Kavitha V, Malarvizhi N, Salomy RM, et al. A study to assess the knowledge, attitude and preventive practices of dengue fever among the people of selected urban slums, Coimbatore. Int J Nurs Educ Res. 2020; 8(1): 35-40. doi: 10.5958/2454-2660.2020.00007.1.

3.      Harapan H, Michie A, Sasmono RT, et al. Dengue : a minireview. Viruses. 2020; 12 (829): 1–35. doi: 10.3390/v12080829.

4.      Kularatne SA, Dalugama C. Dengue infection: global importance, immunopathology and management. Clin Med J R Coll Physicians London. 2022; 22(1): 9–13. doi: 10.7861/clinmed.2021-0791.

5.      Dehghani R, Kassiri H. A review on epidemiology of dengue viral infection as an emerging disease. Res J Pharm Technol. 2021; 14(4): 2296–2301.doi: 10.52711/0974-360X.2021.00406.

6.      D. Bagul P, N. Badar C, J. Tiwari K. Zika virus: a review. Res J Pharmacol Pharmacodyn. 2022; 14(3): 171–173. doi: 10.52711/2321-5836.2022.00029.

7.      Vandali V. Zika virus: a review. Int J Adv Nurs Manag. 2016; 4(2): 167–168. doi: 10.5958/2454-2652.2016.00038.x.

8.      Soonwera M, Moungthipmalai T, Aungtikun J, et al. Combinations of plant essential oils and their major compositions inducing mortality and morphological abnormality of Aedes aegypti and Aedes albopictus. Heliyon. 2022; 8(5): 1-16. doi: 10.1016/j.heliyon.2022.e09346.

9.      Sudaryanto A, Ainnurriza US, Supratman, et al. Mapping the prevalence of dengue fever in Sragen regency Indonesia. Bali Med J. 2021; 10(3): 1107–1110. doi: 10.15562/bmj.v10i3.2821.

10.   Suwantika AA, Kautsar AP, Supadmi W, et al. Cost-effectiveness of dengue vaccination in Indonesia: Considering integrated programs with wolbachia-infected mosquitos and health education. Int J Environ Res Public Health. 2020; 17 (12): 1–15. doi: 10.3390/ijerph17124217.

11.   Hendron RWS, Bonsall MB. The interplay of vaccination and vector control on small dengue networks. J Theor Biol. 2016; 407: 349–361.doi: 10.1016/j.jtbi.2016.07.034.

12.   Dabashini Devi L. Knowledge regarding selected mosquito borne disease and its prevention. Asian J Nurs Educ Res. 2022; 12 (4): 409–412. doi: 10.52711/2349-2996.2022.00087.

13.   Shinde SS, Frew. Dengue fever: A review. Int J Adv Nur Manag. 2016; 4(2): 161–163. doi: 10.5958/2454-2652.2016.00036.6

14.   Aldar S, Deshmukh G. Mosquito repellent, prevention is better than cure. Asian J Res Pharm Sci. 2019; 9(3): 193-198. doi: 10.5958/2231-5659.2019.00030.4

15.   Unissa R, Jyothirmayi B, Mounica A, et al. Dengue. Asian J Res Pharm Sci. 2018; 8(4): 185–191. doi: 10.5958/2231-5659.2018.00032.2.

16.   Nisa K, Taha RM, Nasir S. Factors affecting community’s behavior in using temephos in Banjarmasin city. Int J Sci Basic Appl Res. 2015; 22 (2): 363–374.

17.   Lesmana SD, Maryanti E, Susanty E, et al. Organophosphate resistance in Aedes aegypti: study from dengue hemorrhagic fever endemic subdistrict in Riau, Indonesia. Reports Biochem Mol Biol. 2021; 10 (4): 589–596. doi: 10.52547/rbmb.10.4.589.

18.   Palomino M, Pinto J, Yaρez P, et al. First national-scale evaluation of temephos resistance in Aedes aegypti in Peru. Parasites and Vectors. 2022; 15 (1): 1–13. doi: 10.1186/s13071-022-05310-x.

19.   Corte R La, Melo VAD, Dolabella SS, et al. Variation in temephos resistance in field populations of Aedes aegypti (Diptera: Culicidae) in the state of Sergipe, Northeast Brazil. Rev Soc Bras Med Trop. 2018; 51 (3): 284–290. doi:10.1590/0037-8682-0449-2017.

20.   Valle D, Bellinato DF, Viana-Medeiros PF, et al. Resistance to temephos and deltamethrin in Aedes aegypti from Brazil between 1985 and 2017. Mem Inst Oswaldo Cruz. 2019; 11(3): 1–17. doi: 10.1590/0074-02760180544.

21.   Sudjarwo SA, Ngadino, Koerniasari, et al. Larvicidal Activity of ethanol leaf extract of Pinus merkusii on Aedes aegypti larvae. Res J Pharm Technol. 2017; 10(4): 1011. doi: 10.5958/0974-360x.2017.00182.2.

22.   Johnson AD, Singh A. Toxic effect of biologically active compound rutin extracted from euphorbious plant Codiaeum variegatum against mosquito Culex quinquefasciatus (diptera: culicidae) larvae. Res J Sci Technol. 2017; 9(3): 301–307. doi: 10.5958/2349-2988.2017.00054.7.

23.   Agouillal F, M. Taher Z, Moghrani H, et al. A review of genetic taxonomy, biomolecules chemistry and bioactivities of Citrus hystrix DC. Biosci Biotechnol Res Asia. 2017; 14(1): 285–305. doi: 10.13005/bbra/2446.

24.   Adrianto H, Yotopranoto S, Hamidah. Effectivity of kaffir lime (Citrus hystrix), nasnaran mandarin (Citrus amblycarpa), and Pomelo (Citrus maxima) leaf extract against Aedes aegypti larvae. J Vector-borne Dis Stud. 2014; 6(1): 1–6.doi: https://dx.doi.org/10.22435/aspirator.v6i1.3516.1-6.

25.   Loh FS, Awang RM, Omar D, et al. Insecticidal properties of Citrus hystrix DC leaves essential oil against Spodoptera litura fabricius. J Med Plants Res. 2011; 5(16): 3739–3744.

26.   Husni E, Putri US, Dachriyanus. Chemical content profile of essential oil from kaffir lime (Citrus hystrix DC.) in Tanah Datar regency and antibacterial activity. Proc 2nd Int Conf Contemp Sci Clin Pharm 2021 (ICCSCP 2021). 2022; 40: 174–181.doi: 10.2991/ahsr.k.211105.025.

27.   Janeh M, Osman D, Kambris Z. Damage-induced cell regeneration in the midgut of Aedes albopictus mosquitoes. Sci Rep. 2017; 7: 1–10.doi: 10.1038/srep44594.

28.   Kadu SG. Sublethal effects of indigenous plant extracts on the biochemical composition of midgut of Carpenter ant, Camponotus compressus F. (hymenoptera: formicidae). Adv Zool Bot. 2021; 9(2): 52–59. doi. 10.13189/azb.2021.090203.

29.   Mishra M, Gupta KK, Kumar S. Impact of the stem extract of Thevetia neriifolia on the feeding potential and histological architecture of the midgut epithelial tissue of early fourth instars of Helicoverpa armigera Hubner. Int J Insect Sci. 2015; 7: 53-60. doi: 10.4137/ijis.s29127.

30.   Sharma A, Kumar S, Tripathi P. Effects of Achyranthes aspera extracts on the survival and midgut histo-architecture of Aedes aegypti L. early IV instars. Open Parasitol J. 2018; 6 (1): 41–51. doi: 10.2174/1874421401806010041.

31.   Sarma R, Khanikor B, Mahanta S. Essential oil from Citrus grandis (Sapindales : Rutaceae) as insecticide against Aedes aegypti (L) (Diptera : Culicidae). Int J Mosq Res. 2017; 4(3): 88–92.

32.   Wikandari RJ, Surati S. Effect of Citrus hystrix DC peels extract against morphology and histology of Aedes aegypti. Aspirator - J Vector-borne Dis Stud. 2018; 10(2): 119–126. doi: 10.22435/asp.v10i2.193.

33.   Senthil-Nathan S. A review of resistance mechanisms of synthetic insecticides and botanicals, phytochemicals, and essential oils as alternative larvicidal agents against mosquitoes. Front Physiol. 2020; 10(1591): 1–21. doi: 10.3389/fphys.2019.01591.

34.   Rohmah EA, Subekti S, Rudyanto M. Larvicidal activity and histopathological effect of Averrhoa bilimbi fruit extract on Aedes aegypti from Surabaya, Indonesia. J Parasitol Res. 2020; 2020: 1–5. doi: 10.1155/2020/8866373.

35.   Liu J, Fernandez D, Gao Y, et al. Enzymology, histological and ultrastructural effects of ar-turmerone on Culex pipiens pallens larvae. Insects. 2020; 11(336): 1–13. doi: 10.3390/insects11060336.

36.   Dutra KA, Wanderley Teixeira V, Cruz GS, et al. Morphological and immunohistochemical study of the midgut and fat body of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: noctuidae) treated with essential oils of the genus Piper. Biotech Histochem 2019; 94(7): 498–513. doi: 10.1080/10520295.2019.1599144.

37.   Agwunobi DO, Hu Y, Yu Z, et al. Cymbopogon citratus essential oil-induced ultrastructural & morphological changes in the midgut, cuticle & Haller’s organ of the tick Haemaphysalis longicornis (Acari: Ixodidae). Syst Appl Acarol. 2020; 25(11): 2047–2062. doi: 10.11158/saa.25.11.10.

38.   Al-Mekhlafi FA. Larvicidal, ovicidal activities and histopathological alterations induced by Carum copticum (Apiaceae) extract against Culex pipiens (Diptera: Culicidae). Saudi J Biol Sci. 2018; 25(1): 52–56. doi:10.1016/j.sjbs.2017.02.010.

39.   Tamilventhan A, Jayaprakash A. Larvicidal activity of Terminalia arjuna bark extracts on dengue fever mosquito Aedes aegypti. Res J Pharm Technol. 2019; 12(1): 87–92. doi: 10.5958/0974-360X.2019.00017.9.

40.   Sina I, Zaharah, Sabri MSM. Larvicidal activities of extract flower Averrhoa bilimbi L. towards important species mosquito, Anopheles barbirostris (diptera: Culicidae). Int J Zool Res. 2016; 12(1-2): 25–31.doi: 10.3923/ijzr.2016.25.31.

 

 

 

 

 

Received on 17.03.2023            Modified on 11.05.2023

Accepted on 28.06.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(3):1346-1351.

DOI: 10.52711/0974-360X.2024.00212