Keywords:

Pollinators

Cocoa

Fertilization

Natural pollination

Assisted pollination

Food substrate

Ceratopogonidae: their role in pollination and fertilization at various technological levels of

Theobroma cacao L. production

Ceratopogonidae: su rol en la polinización y fecundación en varios niveles tecnológicos de producción

de Theobroma cacao L.

Ceratopogonidae: seu papel na polinização e fertilização em diversos níveis tecnológicos de produção

de Theobroma cacao L.

Silvia Lorena Montero-Cedeño

Ernesto Gonzalo Cañarte-Bermudez

José Bernardo Navarrete-Cedeño

Antonio Pinargote-Borrero

Pedro Sanchez-Hernández

Rev. Fac. Agron. (LUZ). 2022, 39(3): e223943

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n3.09

Crop Production

Associate editor: Dr. Francisco Osorio-Acosta

Colegio de Postgraduados Campus

Veracruz, Mexico Tepetates, Veracruz,

Escuela Superior Politécnica Agropecuaria de Manabí. Av.

10 de agosto y Granda Centeno. Calceta, Ecuador.

Instituto Nacional de Investigaciones Agropecuarias.

Estación Experimental Portoviejo, Manabí, Ecuador.

Instituto Nacional de Investigaciones Agrícolas, Caracas-

Venezuela.

Received: 09-02-2022

Accepted: 10-07-2022

Published: 25-08-2022

Abstract

The goal of this study was to evaluate the role of pollinators on

the pollination, fertilization and fruit set process in three technological

production levels and three reproduction substrates, determinants in cocoa

yield. Two pollination techniques (natural and assisted) were compared and

as a control, owers were isolated with antiaphid. Twenty-seven observation

units were formed, with three biological replicates. A known number of

owers were evaluated 6, 14, 21 and 36 days after the opening of each ower

on marked branches. The number of active owers, pollinated, fertilized and

fruits formed was recorded. Pollinators present in experimental units were

collected using yellow sticky cards, then taken to the lab and identied.

Pollination occurred up to three days after opening, the ower that remained

with the ovary swollen and attached to the branch/stem was considered

pollinated. Fertilization was recorded fourteen days after ower opening, fruit

set begins after fertilization, an event that was quantied twenty-one days

after ower opening. The systems studied did not inuence the percentage

of pollination, fertilization and fruit formation. However, the substrates did

inuence pollination, fertilization and fruit set. The assisted pollination

technique is signicantly superior to natural pollination. The same species of

Ceratopogonidae were reported at all three levels and substrates, with genera

Forcipomyia and Dasyhelea being the most abundant.

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Rev. Fac. Agron. (LUZ). 2022, 39(3): e223943. July - September. ISSN 2477-9407.2-6 |

Resumen

A objeto de valorar el rol de los polinizadores sobre el proceso

de la polinización, fecundación y formación de frutos en tres

niveles tecnológicos de producción y tres sustratos de reproducción,

determinantes en el rendimiento de cacao. Se compararon dos

técnicas de polinización (natural y asistida) y como control, se

aislaron ores con mangas antiádo. Se conformaron 27 unidades

de observación, con tres réplicas biológicas. Se partió de un número

conocido de ores y se evaluó a los 6, 14, 21 y 36 días después de

la apertura de cada or en ramas marcadas. Se registró el número

de ores activas, polinizadas, fecundadas y frutos formados. Usando

tarjetas pegantes amarillas, se colectó e identicó los polinizadores

presentes en unidades experimentales. La polinización, ocurre

hasta los tres días de la apertura, considerándose polinizada la or

que permaneció con el ovario hinchado, adherida a la rama/tronco.

La fecundación, se registró a los catorce días de la apertura de la

or, la formación de frutos comienza desde la fecundación, evento

que se cuanticó a los veintiún días después de la apertura de las

ores. Los sistemas estudiados no inuyeron en el porcentaje de

polinización, fecundación y formación de frutos. Sin embargo, los

sustratos sí inuyeron en la polinización, fecundación y formación

de frutos. La técnica de polinización asistida es signicativamente

superior a la polinización natural. Se reportaron las mismas especies

de Ceratopogonidae en los tres niveles y sustratos, destacando los

géneros Forcipomyia y Dasyhelea.

Palabras clave: polinizadores, cacao, fecundación, polinización

natural, polinización asistida, sustrato alimenticio.

Resumo

Com o objetivo de avaliar o papel dos polinizadores no

processo de polinização, fertilização e formação dos frutos em três

níveis tecnológicos de produção e três substratos de reprodução,

fatores determinantes na produtividade do cacau. Duas técnicas de

polinização (natural e assistida) foram comparadas e como controle,

ores com mangas anti-afídeos foram isoladas. Foram formadas 27

unidades de observação, com três réplicas biológicas. Foi iniciada

a partir de um número conhecido de ores e avaliada aos 6, 14,

21 e 36 dias após a abertura de cada or em ramos marcados. Foi

registrado o número de ores ativas, polinizadas, fertilizadas e frutos

formados. Por meio de cartões amarelos adesivos, os polinizadores

presentes nas unidades experimentais foram coletados e identicados.

A polinização ocorre até três dias após a abertura, considerando-se

polinizada a or que permaneceu com o ovário inchado, aderido

ao galho / tronco. A fecundação foi registrada quatorze dias após a

abertura da or, a formação dos frutos começa a partir da fecundação,

evento que foi quanticado em vinte e um dia após a abertura das

ores. Os sistemas estudados não inuenciaram na porcentagem

de polinização, fertilização e formação de frutos. No entanto, os

substratos inuenciaram a polinização, fertilização e formação dos

frutos. A técnica de polinização assistida é signicativamente superior

à polinização natural. As mesmas espécies de Ceratopogonidae foram

relatadas nos três níveis e substratos, com destaque para os gêneros

Forcipomyia e Dasyhelea.

Palavras-chave: cacau, fertilização, polinização natural, polinização

assistida, substrato alimentar.

Introduction

Theobroma cacao L. is an important crop worldwide and its

production depends on the activity of pollinating insects. Ninety

percent of the crop requires adequate pollination (FAO, 2008).

This is basically entomophilous, due to the size, arrangement of the

perianth and the condition of incompatibility. Therefore, the presence

and activity of pollinators is essential, especially Diptera of the

Ceratopogonidae family, mostly species of the genus Forcipomyia.

These diptera transfer pollen from a ower of a donor tree (father)

to a ower of a recipient tree (mother), thus effecting pollination,

fertilization and subsequent fruit formation (Córdoba et al., 2013).

In cocoa, fruit production is affected by the reduction of pollinator

populations in their ecosystems (Adjaloo et al., 2012; FAO, 2008;

Salazar-Díaz and Torres-Coto, 2017).

The virtual conservation of the biodiversity of the original forest

is a benet offered by traditional cocoa agroforestry systems at the

ecological level, due to the high diversity and complex structure of

the shade canopy, in which farmers incorporate various tree species,

especially forest and fruit trees, from which they obtain different

products that support the economy of the production units (Gebauer

et al., 2002).

The diversity and abundance of pollinating insects in cocoa

agroforestry systems can be inuenced by several biotic and abiotic

factors, as well as vegetation cover, organic matter, spacing of

shade trees and shade tree owering (Adjaloo et al., 2012; Ramos-

Serrano, 2011; Schroth and Harvey, 2007). Habitat suitability

for Ceratopogonidae species require shaded conditions, balanced

humidity, and rich organic matter to develop and thrive (Kaufmann,

1975; Toledo-Hernández et al., 2017; Young, 1982, 1986).

The environmental crisis of the world is observed in threats to

environmental goods and services, loss of natural vegetation, soil

degradation, ecosystem degradation, environmental pollution, as

well as climate change and the accelerated disappearance of species

(Plan Nacional de Desarrollo, 2017). Hence, this research aims to

assess the activity of pollinators on pollination, fertilization and fruit

formation of cocoa at different technological production levels and

three reproduction substrates determinants in the yield of ne cocoa.

Materials and methods

This research was carried out at the Portoviejo Experimental Station

of INIAP, located in Santa Ana-Manabí, Ecuador. Geographically

located (1°07’25.7 “S and 80°24’50.9 “W) and conditions (81% RH;

851.57 mm and 26.4 °C).

Experimental management

The work was carried out at three technological production

levels and three reproduction substrates (cocoa husks, leaf litter and

musaceae pseudostem). The monoculture level corresponded to a

25-year-old plantation with three known clones (EEP 95, EEP 96

and EPP 103); the cocoa/fruit level consisted of a 25-year-old cocoa

plantation associated with three citrus species (Citrus reshni Hort. ex

Tan, Citrus reticulata Blanco, Citrus sinensis L. Osbeck). The cocoa/

forest level consisted of cocoa with timber trees of the Guayaquil

yellow species Handroanthus chrysanthus (Jacq.) (Bignoniaceae) y

Cedrus atlantica (Endl.) Manetti ex Carrière (Pinaceae), 25 years

old. For the study, a known number of cocoa owers in each of the

plots (27 observation units) was used as the starting point. Three

biological replicates were carried out. An average of 8.3, 7.6 and 10.7

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Montero-Cedeño et al. Rev. Fac. Agron. (LUZ). 2022, 39(3): e2239433-6 |

owers were used for biological replicates I, II and III, respectively.

Evaluations were made at 3, 6, 14, 14, 21 and 36 days after ower

opening. Two pollination techniques (natural and assisted) were

compared and isolated owers in sleeves made with anti-aphid mesh

free from pollinator interference were used as controls.

For the implementation of pollination techniques in each of the

production levels, the methodology described by Córdoba et al.

(2013) was used as a reference. In each plot, those trees with more

owers were considered, and in each selected tree, three branches

(observations) were chosen for each technique. First, in each of

the selected branches, all open, dry owers and small buds were

eliminated, leaving only closed mature ower buds, which were

counted. From that date, the number of pollinated owers, fertilized

owers and number of fruits formed were recorded.

For the free or natural pollination technique, the section of the

branch on each marked branch (gure 1A) containing the selected

ower buds, ready to be opened the following morning, was delimited

with tape and set free to be visited by the pollinating midges.

For the manual technique, the ower buds that were close to

opening were selected on each marked branch 24 hours before.

These were covered with cotton to prevent the arrival of pollinators.

Recently opened owers were selected the following morning and

used as pollen donors. Subsequently, the stamens were removed with

ne forceps from each of the selected owers (covered with the vial)

(gure 1B). Finally, pollen from the anthers of the male donor is

gently rubbed onto the surface of the style and stigma of the recipient

ower (Vera and Mogrovejo, 2010).

In order to have a control as in the previous techniques, three

branches per tree (3 trees) containing ower buds were selected,

which were individualized with 60 cm long sleeves, constructed with

anti-aphid mesh. The sleeves were hermetically sealed at the ends with

tape, thus preventing the entrance of any oral visitor (gure 1C). For

the sampling and identication of pollinators of the Ceratopogonidae

family present in the three levels of cocoa production, yellow plastic

traps (5 x 5 cm) with light oil were used (gure 1D). A total of 27

traps were placed for each production level, distributed in nine traps

for each pollination technique, located next to each branch containing

the marked owers. Three days later, they were removed and taken

to the Entomology laboratory of the Portoviejo Station of INIAP

for identication and counting of pollinators, using the reference

collection maintained by the laboratory.

Figure 1. Pollination technique and pollinators: free or natural pollination (A); assisted pollination (B); mesh sleeve control (C); yellow

sticky traps (D).

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Statistical analysis

Variables were analyzed in a variance analysis corresponding to

a split-plot design with three replications. The Wilcoxon and Barlett

tests were used to test normality and homogeneity of variances,

respectively. Tukey’s test at 0.05 was used for dividing means. The

Agricolae package of R Studio software (Mendiburu and Yaseen,

2020) was used for these analyses.

Results and discussion

In the variable percentage of pollinated owers between the

technological levels and reproduction substrates, the ADEVA

did not establish signicant statistical differences (P>0.05)

between treatments in each of the pollination techniques (natural

and assisted), while assisted pollination had higher values in the

monoculture of cocoa (31%) of replicate III and the leaf litter

substrate with 27% of pollinated owers in replicate I. While, in

natural pollination, a maximum of 12% of pollinated owers were

obtained in the fruiting cocoa technological level in replicate II

and 15% in the substrate with pseudostem of musaceae (plantain/

banana) in replicate III. Cocoa owers that were in the sleeves

(control) always presented zero values in the three biological

replicates, both in the technological levels and in the substrates.

The sleeves prevented the access of oral visitors to the cocoa

ower, preventing pollination. These results conrm the role of

Ceratopogonidae on the natural pollination of cocoa owers. The

highest percentage of pollinated owers in monoculture cocoa with

leaf litter in the assisted technique is due to the technique and not to

the action of pollinators, which occurs in lower population in this

substrate. Consequently, the assisted technique was responsible for

the increase in pollinated owers.

These results agree with studies conducted by Forbes and

Northeld (2017), in Australia, who manipulated the habitat

in a monoculture cocoa plantation with articial treatment and

pollination (manual pollination) to evaluate the addition effects of

cocoa shells on pollination, obtaining that cocoa shells increased

the presence of pollinators in the plantations, thus improving the

efciency of pollination and fruit set in cocoa.

The results of the variables percentage of fertilized owers and

average percentage of cocoa fruits formed in the three biological

replicates at 14 and 21 days after ower opening recorded in the two

factors under study, show equal average values in both variables,

i.e., 100% of fertilized owers yielded cocoa fruits. The ADEVA

did not establish signicant statistical differences (P>0.05) for the

technological levels factor in any of the two pollination techniques

(natural and assisted) in the three biological replicates carried out.

While the substrate factor did register signicant differences in the

natural pollination of replicate III (F2.18 = 16.32; P<0.01), where the

substrate with musacea (plantain/banana), stands out signicantly

from cocoa husk and leaf litter (Tukey), for its higher percentage

of fertilized owers and fruits formed (12%) (gure 2). The higher

fertilization percentage and fruit formation may be inuenced by

the presence of pollinators in these plots, where the humidity and

decomposing material provided by the musaceae favors pollinator

populations.

It is known that the immature instars of Ceratopogonidae

develop mainly in moist and decaying plant matter, such as cocoa

cobs, pseudostem of musaceae that offer an excellent substrate for

feeding, since the larvae of these insects feed mainly on bacteria and

fungi that develop in these substrates; as well as habitat and sources

of reproduction and protection of the immature stages of these

pollinators (Adjaloo et al., 2012). This is also stated by Shackelford

et al. (2013), who mention that some pollinators and predators have

shown responses to habitat manipulation and reduced pesticide

use (Bianchi et al., 2006), with positive effects on fruit set and

agricultural productivity. Also, Bianchi et al. (2006) and Wratten

et al. (2012) mention that habitat manipulation promotes pollinator

populations and provides additional benets such as the availability

of habitats and food resources for pollinators and other organisms

present in the agroecosystem, favoring agricultural production and

biological conservation. As for the cocoa owers kept inside the

sleeves (control control), they presented zero fertilized owers in

the three biological replicates, both in the technological levels and

between substrates, because pollination was interrupted by the non-

intervention of the Ceratopogonidae in pollination.

Figure 2. Percentage of cocoa ower fertilization and fruits

formed.

When comparing the percentage of pollinated and fertilized

owers and fruits formed, between the two pollination techniques

(natural and assisted), in the three biological replicates, signicant

statistical differences were found in the variables studied in

most of the biological replicates (P<0.05). The percentage of

pollinated owers, fertilized owers and fruits formed was always

signicantly higher in the assisted pollination technique (gure

3), with an overall average of 20% of pollinated owers, 16% of

fertilized owers and 15% of fruits formed. In contrast with the

natural pollination technique, where 9% of owers were pollinated,

4% fertilized and 4% of fruits formed.

These results agree with those reported by Córdoba et al.

(2013), who state that ower fertilization and fruit formation

of up to 25% can be achieved with manual pollination in cocoa

agroforestry systems in Indonesia, while only 4% of owers are

fertilized with natural pollination. Likewise, Vera and Mogrovejo

(2010) determined that better results are obtained with the assisted

pollination technique, since the number of fruits can be doubled and

therefore productivity can be increased. In addition, the application

of this technique facilitates the management of fruit diseases that

increase during the rainy season. However, Wessel and Quist

(2015), argue that although pollination is improved, the pollination

technique is not sufcient to guarantee fertilization and potential

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Montero-Cedeño et al. Rev. Fac. Agron. (LUZ). 2022, 39(3): e2239435-6 |

yield of cocoa. Thus, there are factors such as self-incompatibility,

crop management and tree aging (>25 years) that can considerably

decrease these yields (Claus et al., 2018). Other studies conducted

by Vera and Vera (2018), indicate that despite not having found

signicant differences between manual and natural technique in

Criollo cocoa plantations, they reported differences between natural

and assisted pollination in CCN-51 cocoa plantations, being higher

in assisted pollination. In addition, Toledo-Hernández et al. (2020)

report that manual pollination in Indonesia increases production up

to 51% if 13% of the tree’s owers are pollinated, and could even

increase up to 161% if 100% of the tree’s owers are pollinated,

which, for the farmer, would be equivalent to a gain of up to 69%.

Figure 3. Percentage of pollinated owers, fertilized owers and

formed cocoa fruits.

Regarding the presence of Ceratopogonidae, ve species were

identied: Forcipomyia (Forcipomyia) quatei Wirth, Forcipomyia

(Forcipomyia) youngi Wirth, Dasyhelea cacaoi Wirth & Waugh,

Dasyhelea borgmeieri Wirth & Waugh, Culicoides pusillus Lutz,

which were present in the three technological production levels and

substrates in the three biological replicates. The genera Forcipomyia

and Dasihelea are widely known as cocoa pollinators (Cañarte-

Bermúdez et al., 2021; Kaufmann, 1975; Montero et al., 2019).

Likewise, these genera found in this research agree with some groups

found by Young (1982), in studies conducted in cocoa plantations,

reporting 22 species of Ceratopogonidae distributed in six genera,

among which were the groups present in our research. Azhar and

Wahi (1984) also reported the species Forcipomyia sp., Dasyhelea

sp., Brachypogon sp. and Atrichopogon sp., coinciding with several

species found in our work. Also, Sánchez et al. (2001), cite species

of Forcipomyia. Martinez et al. (2000) also reported the presence of

Forcipomyia brachyrhynchus.

Out of the ve identied species of adult specimens of the

Ceratopogonidae family, yellow.trap

-1

observed among the

technological levels, ADEVA established signicant statistical

differences for the species F. quatei only in replicate III (F

2.18

= 33.26;

P<0.001), registering the highest average number of specimens/

trap in the cocoa + forest technological level, signicantly different

according to Tukey to the other two technological levels. These

results differ from those reported by Young (1986), who found that

there were different groups of pollinators in two farms evaluated,

indicating that the greater presence of groups of pollinators in one

farm was due to the greater diversity of plant species that provide

heterogeneity in the substrate, as opposed to the other, which was a

simple agroecosystem.

In relation to the breeding substrate, signicant statistical

differences were recorded in three of the ve Ceratopogonidae

species identied (F. quatei, F. youngi and D. borgmeieri).

Regarding F. quatei, analysis of variance reported highly signicant

differences in replicates I and III (F

2,18

= 22.75; P<0.01 and F

2,18

33.26; P<0.01), respectively. In both replicates, the highest average

number of specimens/trap was recorded in the substrate pseudostem

of musaceae (plantain/banana), while the lowest average number of

adult specimens was recorded in the plots with leaf litter substrate.

yellow trap

-1

. In the pollinator species F. youngi, the substrate with

pseudostem of Musaceae showed a higher average value of adult

specimens/trap of this species, conrming the leaf litter as the

substrate that contributes the least to the presence of pollinators.

In the species D. cacaoi, the analysis of variance found signicant

statistical differences in replicate I (F

2.18

= 8.00; P<0.05), presenting

the highest average values of the number of adult specimens/trap in

musaceae pseudostem substrate, while the population was zero in

substrate with leaf litter.

These results are supported by a previous prognosis,

where substrates (plantain pseudostem) for the reproduction of

Ceratopogonidae midges in cocoa plantations increased the presence

of pollinators (Montero et al., 2019). Likewise, Bravo et al. (2010)

state that the suitable variables in the behavior of mosquito populations

are mostly related to the substrate and not to the abundance of

the different species present in the plantations. Kaufmann (1975)

and Young (1982) showed that pollinators frequently oviposit

and complete their cycle in the soil (cocoa leaf litter). In addition,

Forbes and Northeld (2017), stated that decomposing cocoa shells

inuenced the increase of cocoa-pollinating mosquito populations

through increased habitat availability for oviposition and successful

larval development, compared to the control without cocoa shells.

Conclusions

The technological production levels did not inuence pollination,

fertilization and fruit formation in the two pollination techniques.

While the substrates of musaceae pseudostems and cocoa husks

signicantly inuenced the increase pollination, fertilization and

fruit formation in the two pollination techniques. The percentage

of pollinated and fertilized owers and fruits formed, obtained with

the assisted technique was higher than with natural pollination. The

sleeving technique made it possible to verify the role of oral visitors

on the pollination and fertilization of cocoa. The Ceratopogonidae

species identied in this study agree with the diversity of pollinators

cited in the literature, associated with the technological levels of cocoa

production, where the genera Forcipomyia and Dasyhelea stand out.

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