Seeds of Cucurbita maxima and Carica papaya are effective for controlling monogeneans in the gills of Leporinus macrocephalus

Sementes de Cucurbita maxima e Carica papaya são eficazes no controle de monogenéticos das brânquias de Leporinus macrocephalus

Abstract

This study was carried out to evaluate the anthelminthic efficacy of seeds of Cucurbita maxima and Carica papaya for controlling monogeneans in the gills of Leporinus macrocephalus, besides hepatosomatic and splenosomatic index and condition factor of host. The fish were fed with seeds of C. maxima or C. papaya for seven days, and these treatments did not cause any mortality among them. Jainus leporini, Urocleidoides paradoxus, Urocleidoides eremitus and Tereancistrum parvus were the monogeneans found, and their prevalence in fish fed with seeds of C. papaya was 100%, while in fish fed with C. maxima the prevalence was 42.8%. Fish fed with seeds of C. papaya showed decreased in intensity and abundance of monogeneans, while fish fed with seeds of C. maxima presented decreased in abundance. Feeding of L. macrocephalus with seeds of C. maxima or C. papaya had efficacy of 69.6 and 67.8%, respectively. The hepatosomatic index of fish fed with seeds of C. maxima or C. papaya was not affected by the treatments. However, the splenosomatic index and condition factor of fish fed with C. maxima seeds decreased. Seeds of C. maxima and C. papaya may be used for controlling monogeneans of L. macrocephalus in fish farming.

Keywords:
Monogenea; phytotherapy; parasite; treatment

Resumo

Este estudo avaliou a eficácia antihelmíntica de sementes de Cucurbita maxima e Carica papaya no controle de monogenéticos das brânquias de Leporinus macrocephalus, além do índice hepatossomático e esplenossomático e do fator de condição do hospedeiro. Os peixes foram alimentados com sementes de C. maxima ou C. papaya durante sete dias, e os tratamentos não causaram mortalidade. Monogenéticos Jainus leporini, Urocleidoides paradoxus, Urocleidoides eremitus e Tereancistrum parvus tiveram prevalência de 100% nos peixes alimentados com sementes de C. papaya, enquanto nos peixes alimentados com C. maxima a prevalência foi de 42,8%. Os peixes alimentados com sementes de C. papaya apresentaram diminuição na intensidade média e abundância média de monogenéticos, enquanto os peixes alimentados com sementes de C. maxima apresentaram uma diminuição na abundância média. A alimentação de L. macrocephalus com sementes de C. maxima ou C. papaya teve eficácia de 69,6 e 67,8%, respectivamente. O índice hepatossomático dos peixes alimentados com sementes de C. maxima ou C. papaya não foi afetada pelos tratamentos. No entanto, o índice esplenossomático e o fator de condição dos peixes alimentados com sementes de C. maxima diminuíram. Sementes de C. maxima e C. papaya podem ser usadas no controle de monogenéticos das brânquias de L. macrocephalus em piscicultura.

Palavras-chave:
Monogenea; fitoterapia; parasito; tratamento

Introduction

Fish are an important source of protein because of their short production cycle. Fish aquaculture plays an important socioeconomic role around world, including in Brazil. Around one million people are directly employed in fish farming in Brazil, and this generates another two million indirect jobs. In 2021, Brazilian fish farming generated US$ 1.5 billion in revenues (PeixeBR, 2022). However, intensification of fish aquaculture has led to increased levels of diseases due to problems of inadequate management. Infection by Monogenea is one of the diseases thus caused. Controlling parasitic diseases within the fish aquaculture industry helps to raise its economic and socioeconomic levels.

Monogeneans are parasites in the phylum Platyhelminthes that have a short and direct life cycle. Their vertical transmission facilitates infection levels within fish aquaculture (Alves et al., 2019; Cruz et al., 2022). Their pathogenicity is directly associated with organ fixation, infection intensity, feeding strategy and general host health (Cruz et al., 2022). Hence, it is difficult to control these ectoparasites in fish farming.

Many chemical drugs have been used against these parasites, but they all have some drawbacks, e.g., low efficacy, toxicity to hosts and environmental and human health problems (Alves et al., 2019; Cruz et al., 2022). In addition, long-term use of chemical drugs can lead to drug resistance among these parasites (Jeyavani et al., 2022). Hence, increasing attention has been paid to the use of traditional plant-based medicines and their bioactive products (phytotherapy) for controlling parasitic diseases in fish aquaculture (Fujimoto et al., 2012; Tavares-Dias, 2018; Trasviña-Moreno et al., 2019; Zhu, 2020; Jeyavani et al., 2022). Herbal therapy does not significantly pollute the aquaculture environment, and is not toxic to humans, thereby ensuring food safety (Zhu, 2020).

Among the herbal products used as alternative methods for controlling the diseases caused by helminths in different animal species are the seeds of pumpkins (Cucurbita spp.) (Feitosa et al., 2013; Grzybek et al., 2016; Marie-Magdeleine et al., 2009; Lima et al., 2020) and papaya (Carica papaya) (Shaziya & Goyal, 2012; Fujimoto et al., 2012; Feroza et al., 2017; Trasviña-Moreno et al., 2019). Both of these phytotherapeutics are relatively inexpensive alternatives, compared with the currently available chemotherapeutics, and have been considered to be good candidates for providing anthelminthic control.

As there is an urgent need for innovative methods that could act towards controlling the diseases caused by monogeneans in farmed fish, use of both Cucurbita maxima (Fujimoto et al., 2012) and Carica papaya (Fujimoto et al., 2012; Trasviña-Moreno et al., 2019) has been recommended. However, these phytotherapeutics have not been used for control and treatment against monogeneans in Leporinus macrocephalus, a fish that is reared in Brazilian aquaculture. Thus, the aim of the present study was to investigate the anthelminthic efficacy of C. maxima and C. papaya seeds for controlling monogeneans in the gills of L. macrocephalus, as well as hepatosomatic index, splenosomatic index and condition factor of host.

Materials and Methods

Fish and monogenean parasites

One hundred fingerlings of L. macrocephalus were obtained from a commercial fish farm in Rio Branco, in the state of Acre, Brazil, and were kept at the laboratory of the Instituto Federal do Acre (IFAC), Campus Baixada do Sol, in Rio Branco, Brazil. The fish were acclimatized for 10 days in a 1,000 L tank, with constant water flow and aeration, and were fed twice a day with commercial feed containing 35% crude protein (Guabi, Brazil). The following water parameters were maintained in the tanks: temperature at 29.1 ± 0.1 °C, dissolved oxygen at 5.6 ± 0.2 mg/L, pH at 5.4 ± 0.2, total ammonia at 0.4 ± 0.01 mg/L, alkalinity at 12.0 ± 0.1 mg/L and water hardness at 12.0 ± 0.1 mg/L. The fish excrement and feed residues accumulated in the bottom of the tanks was removed once every day. This stock of fish was used in all the in vivo assays.

The monogeneans used in these experiments were obtained from naturally infested fish.

Obtaining and preparing of Cucurbita maxima and Carica papaya seeds

Cucurbita maxima and C. papaya were acquired from commercial sources in the city of Rio Branco, state of Acre, Brazil. The seeds of C. maxima and C. papaya were removed and dried in an oven at 50 °C for 24 h. Subsequently, these seeds were crushed using a meat grinder to produce particles of sizes around 6 mm.

Feeding with seeds of Cucurbita maxima and Carica papaya

Fifty-four fingerlings of L. macrocephalus (6.9 ± 1.4 cm and 4.6 ± 3.8 g) that were naturally parasitized by monogeneans were randomly distributed into nine 250 L tanks. They were kept in a static water system under constant aeration for seven days. The following water parameters were maintained in the tanks: temperature at 30.1 ± 0.1 °C, dissolved oxygen at 5.5 ± 0.2 m /L, pH at 5.6 ± 0.2, total ammonia at 0.4 ± 0.01 mg/L, alkalinity at 11.0 ± 0.1 mg/L and water hardness at 11.0 ± 0.1 mg/L. The fish excrement and feed residues accumulated in the bottom of the tanks was removed once every day.

Control fish were fed once a day ad libitum with commercial feed containing 35% crude protein (Guabi, Brazil), for seven days. This control group consisted of three replicates with six fish each (18 fish per treatment). One group with three replicates of six fish each (18 fish per treatment) was fed once a day ad libitum with crushed seeds of C. maxima, for seven days. Another group with three replicates of six fish each (18 fish per treatment) was fed once a day ad libitum with crushed seeds of C. papaya, for seven days. The leftovers of crushed seeds were removed from the tanks every day.

After seven days of feeding with crushed seeds of C. maxima and C. papaya, the fish were euthanized by means of medullary section, weighed (g) and measured (cm). Their gills were excised, fixed in 5% formalin and examined under a stereomicroscope to identify and quantify the monogenean parasites. The parasites were prepared for identification as recommended by Eiras et al. (2006). After parasite quantification, the prevalence and mean intensity of infection were calculated as described by Bush et al. (1997) and the efficacy of each treatment as described by Fujimoto et al. (2012). Liver and spleen weight were measured and were used to determine the splenosomatic index (SSI) and hepatosomatic index (HSI) of fish (Tavares-Dias et al., 2000). Body weight and length were used to determine the relative condition factor (Kn) (Le Cren, 1951).

The data were evaluated based on the Shapiro–Wilk normality test and Bartlett’s test of homoscedasticity. Because the intensity and abundance data were not normally distributed, they were analyzed by the Kruskal–Wallis test, followed by Dunn’s test for comparison among medians (Zar, 2010).

This study was developed in accordance with the principles adopted by the Brazilian College of Animal Experimentation (COBEA) and with authorization from the Ethics Committee in the Use of Animals of Embrapa Amapá (Protocol No. 013-CEUA/CPAFAP).

Results and Discussion

The hepatosomatic index, length and weight of L. macrocephalus fed with seeds of C. maxima or C. papaya for seven days were not affected by the treatments. However, the splenosomatic index and condition factor of L. macrocephalus fed with C. maxima seeds decreased (Table 1). The hepatosomatic index is an indirect measurement of glycogen and carbohydrate levels, and can be used to indicate the nutritional state of the fish. The splenosomatic index is a measurement of both the immune status and the hematopoietic capacity of the fish (Tavares-Dias et al., 2000; Voorhees et al., 2019). Therefore, the results from L. macrocephalus indicated that the growth and body condition was negatively influenced probably by the reduction in ingestion of food, and also by the immune status and hematopoiesis.

Table 1
Infection rates by monogeneans and body parameters of Leporinus macrocephalus fed with seeds of Cucurbita maxima and Carica papaya during seven days.

Parameters	Control	Carica papaya seed	Cucurbita maxima seed
Length (cm)	6.3 ± 1.0a	7.6 ± 1.4a	7.2 ± 1.6a
Weight (g)	3.1 ± 2.5b	6.3 ± 4.3b	5.1 ± 4.9b
Prevalence (%)	100	100	42.8
Mean intensity	12.1 ± 14.3a	3.3 ± 2.1b	8.2 ± 12.6ab
Mean abundance	12.1 ± 14.3a	3.3 ± 2.1b	3.9 ± 9.2b
HSI (%)	1.3 ± 0.3a	1.3 ± 0.3a	1.1 ± 0.4a
SSI (%)	0.9 ± 1.1a	0.7 ± 0.7a	0.3 ± 0.2b
Kn	0.90 ± 1.16a	1.01 ± 0.31a	0.69 ± 0.40b

• Values express mean ± Standard deviation. Different letters in same line indicate differences by the Dunn test (p>0.05). SSI: Splenosomatic index, HIS: Hepatosomatic index, Kn: Relative condition factor.

In the present study, none of the treatments with diets containing seeds of C. maxima or C. papaya caused any mortality of L. macrocephalus over the seven-day period. The Leporinus macrocephalus specimens were naturally infected by Jainus leporini, Urocleidoides paradoxus, Urocleidoides eremitus and Tereancistrum parvus, which showed prevalence of 100% among the fish fed with seeds of C. papaya for seven days, while among the fish fed with C. maxima the prevalence was lower. In addition, the fish fed with seeds of C. papaya showed lower mean intensity and mean abundance of monogeneans, while the fish fed with seeds of C. maxima presented lower mean abundance of monogeneans (Table 1).

Disease management and control are difficult, particularly because of the limited availability of low-cost licensed drugs with proven efficacy (Alves et al., 2019). Furthermore, drugs need to be safe and effective, and therapeutic agents must also present low toxicity when used to treated fish. Feeding of L. macrocephalus with seeds of C. maxima or C. papaya, for seven days, showed efficacy of 69.6 and 67.8%, respectively (Figure 1). Similar results were previously reported with regard to the efficacy of treatments against monogeneans in Astyanax zonatus (72%), after these fish were fed with seeds of C. papaya for seven days. This contrasted with other results from C. maxima, in which the efficacy was shown to only be 39% (Fujimoto et al., 2012). Water-ethanol extracts of C. papaya did not show antiparasitic properties against Neobenedenia sp. in Seriola lalandi, probably because of instability or low concentration of the bioactive compounds used (Trasviña-Moreno et al., 2019).

Figure 1
Antiparasitic efficacy of Cucurbita maxima and Carica papaya seeds against monogeneans of gills of Leporinus macrocephalus.

The anthelmintic activity of C. papaya seeds has been attributed to the compound benzyl isothiocyanate, along with presence of carpaine and carpasemine (Kermanshai et al., 2001; Krishna et al., 2008; Trasviña-Moreno et al., 2019). It is believed that the anthelmintic activity of the seeds of Cucurbita spp. is due to cucurbitacin B, cucurbitin (3-amino-pyrrolidine-3-carboxylic acid), saponins and sterols, but roles for other compounds such as cucurmosin, berberine and palmatine possibly cannot be ruled out (Marie-Magdeleine et al., 2009; Grzybek et al., 2016).

In conclusion, our results demonstrated that C. maxima and C. papaya seeds were effective for controlling monogeneans in the gills of L. macrocephalus. Therefore, their use may be signaled as an alternative anthelminthic for controlling and treating monogenean infections in this fish in fish farming.

Acknowledgements

Marcos Tavares-Dias was supported (Grant 303013/2015-0) by research fellowship from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq, Brazil). Tiago Araújo Lima and Vitória Moura Carvalho received PIBTI fellowship from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico, and Eliane Xavier Souza received research fellowship from Instituto Federal do Acre (IFAC, Brazil).

• How to cite: Negreiros LP, Carvalho VM, Lima TA, Sousa EX, Tavares-Dias M. Seeds of Cucurbita maxima and Carica papaya are effective for controlling monogeneans in the gills of Leporinus macrocephalus. Braz J Vet Parasitol 2022; 31(2): e006822. https://doi.org/10.1590/S1984-29612022029

References

• Alves CMG, Nogueira JN, Barriga IB, Santos JR, Santos GG, Tavares-Dias M. Albendazole, levamisole and ivermectin are effective against monogeneans of Colossoma macropomum (Pisces: serrasalmidae). J Fish Dis 2019; 42(3): 405-412. http://dx.doi.org/10.1111/jfd.12952 PMid:30659617.
  » http://dx.doi.org/10.1111/jfd.12952
• Associação Brasileira da Piscicultura – PEIXEBR. Anuário Peixe BR de piscicultura 2021 [online]. 2022 [cited 2022 Apr 20]. Available form: https://www.peixebr.com.br/anuario2022
  » https://www.peixebr.com.br/anuario2022
• Bush AO, Lafferty KD, Lotz JM, Shostak W. Parasitology meets ecology on its own terms: Margolis et al. revisited. J Parasitol 1997; 83(4): 575-583. http://dx.doi.org/10.2307/3284227 PMid:9267395.
  » http://dx.doi.org/10.2307/3284227
• Cruz MG, Jerônimo GT, Bentes SPC, Gonçalves LU. Trichlorfon is effective against Dawestrema cycloancistrium and does not alter the physiological parameters of arapaima (Arapaima gigas): a large Neotropical fish from the Amazon. J Fish Dis 2022; 45(1): 203-212. http://dx.doi.org/10.1111/jfd.13549 PMid:34779526.
  » http://dx.doi.org/10.1111/jfd.13549
• Eiras JC, Takemoto RM, Pavanelli GC. Métodos de estudos e técnicas laboratóriais em parasitologia de peixes Maringá: Eduem; 2006.
• Feitosa TF, Vilela VLR, Athayde ACR, Braga FR, Dantas ES, Vieira VD, et al. Anthelmintic efficacy of pumpkin seed (Cucurbita pepo Linnaeus, 1753) on ostrich gastrointestinal nematodes in a semiarid region of Paraíba State, Brazil. Trop Anim Health Prod 2013; 45(1): 123-127. http://dx.doi.org/10.1007/s11250-012-0182-5 PMid:22684690.
  » http://dx.doi.org/10.1007/s11250-012-0182-5
• Feroza S, Arijo AG, Zahid IR. Effect of papaya and neem seeds on Ascaridia galli infection in broiler Chicken. Pak J Nematol 2017; 35(1): 105-111. http://dx.doi.org/10.18681/pjn.v35.i01.p105-111
  » http://dx.doi.org/10.18681/pjn.v35.i01.p105-111
• Fujimoto TY, Costa HC, Ramos FM. Controle alternativo de helmintos de Astyanax cf. zonatus utilizando fitoterapia com sementes de abóbora (Cucurbita maxima) e mamão (Carica papaya). Pesq Vet Bras 2012; 32(1): 5-10. http://dx.doi.org/10.1590/S0100-736X2012000100002
  » http://dx.doi.org/10.1590/S0100-736X2012000100002
• Grzybek M, Kukula-Koch W, Strachecka A, Jaworska A, Phiri AM, Paleolog J, et al. Evaluation of anthelmintic activity and composition of Pumpkin (Cucurbita pepo L.) seed extracts - In vitro and in vivo studies. Int J Mol Sci 2016; 17(9): 1456. http://dx.doi.org/10.3390/ijms17091456 PMid:27598135.
  » http://dx.doi.org/10.3390/ijms17091456
• Jeyavani J, Sibiya A, Sivakamavalli J, Divya M, Preetham E, Vaseeharan B, et al. Phytotherapy and combined nanoformulations as a promising disease management in aquaculture: a review. Aquacult Int 2022; 30(2): 1071-1086. http://dx.doi.org/10.1007/s10499-022-00848-0
  » http://dx.doi.org/10.1007/s10499-022-00848-0
• Kermanshai R, McCarry BE, Rosenfeld J, Summers PS, Weretilnyk EA, Sorger GJ. Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts. Phytochemistry 2001; 57(3): 427-435. http://dx.doi.org/10.1016/S0031-9422(01)00077-2 PMid:11393524.
  » http://dx.doi.org/10.1016/S0031-9422(01)00077-2
• Krishna KL, Paridhavi M, Patel JA. Review on nutritional, medicinal and pharmacological properties of papya (Carica papaya Linn.). Nat Prod Radiance 2008; 7(4): 364-373.
• Le Cren ED. The length-weight relationship and seasonal cycle in gonad weight and condition in the perch (Perca fluviatilis). J Anim Ecol 1951; 20(2): 201-219. http://dx.doi.org/10.2307/1540
  » http://dx.doi.org/10.2307/1540
• Lima DF, Brainer MMA, Fabino RF, Silva BC, Godoy MM, Fabino Neto R, et al. Potencial antihelmíntico de sementes de abóbora (Cucurbita mochata) em equinos. Braz J Anim Environ Res 2020; 3(3): 952-965. http://dx.doi.org/10.34188/bjaerv3n3-016
  » http://dx.doi.org/10.34188/bjaerv3n3-016
• Marie-Magdeleine C, Hoste H, Mahieu M, Varo H, Archimede H. In vitro effects of Cucurbita moschata seed extracts on Haemonchus contortus. Vet Parasitol 2009; 161(1-2): 99-105. http://dx.doi.org/10.1016/j.vetpar.2008.12.008 PMid:19135803.
  » http://dx.doi.org/10.1016/j.vetpar.2008.12.008
• Shaziya BI, Goyal PK. Anthelmintic effect of natural plant (Carica papaya) extract against the gastrointestinal nematode, Ancylostoma caninum in mice. Int Res J Biol Sci 2012; 1(1): 2-6.
• Tavares-Dias M, Martins ML, Moraes FR. Relação hepatosomática e esplenosomática em peixes teleósteos de cultivo. Rev Bras Zool 2000; 17(1): 273-281. http://dx.doi.org/10.1590/S0101-81752000000100024
  » http://dx.doi.org/10.1590/S0101-81752000000100024
• Tavares-Dias M. Current knowledge on use of essential oils as alternative treatment against fish parasites. Aquat Living Resour 2018; 31: 13. http://dx.doi.org/10.1051/alr/2018001
  » http://dx.doi.org/10.1051/alr/2018001
• Trasviña-Moreno AG, Ascencio F, Angulo C, Hutson KS, Avilés-Quevedo A, Inohuye-Rivera RB, et al. Plant extracts as a natural treatment against the fish ectoparasite Neobenedenia sp. (Monogenea: Capsalidae). J Helminthol 2019; 93(1): 57-65. http://dx.doi.org/10.1017/S0022149X17001122 PMid:29248015.
  » http://dx.doi.org/10.1017/S0022149X17001122
• Voorhees JM, Barnes ME, Chipps SR, Brown ML. Bioprocessed soybean meal replacement of fish meal in rainbow trout (Oncorhynchus mykiss) diets. Cogent Food Agric 2019; 5(1): 1579482. http://dx.doi.org/10.1080/23311932.2019.1579482
  » http://dx.doi.org/10.1080/23311932.2019.1579482
• Zar JH. Biostatistical analysis 5th ed. New Jersey: Prentice-Hall; 2010.
• Zhu F. A review on the application of herbal medicines in the disease control of aquatic animals. Aquaculture 2020; 526:735422. https://doi.org/10.1016/j.aquaculture.2020.735422
  » https://doi.org/10.1016/j.aquaculture.2020.735422

Publication Dates

• Publication in this collection
  06 June 2022
• Date of issue
  2022

History

• Received
  03 May 2022
• Accepted
  13 May 2022