SciELO - Scientific Electronic Library Online

 
vol.29 número1Compatibilidad de la púa y el portainjerto en Pinus patula Schiede ex Schltdl. & Cham. como respuesta a la variación genotípica índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

  • No hay artículos similaresSimilares en SciELO

Compartir


Revista Chapingo serie ciencias forestales y del ambiente

versión On-line ISSN 2007-4018versión impresa ISSN 2007-3828

Rev. Chapingo ser. cienc. for. ambient vol.29 no.1 Chapingo ene./abr. 2023  Epub 23-Jun-2024

https://doi.org/10.5154/r.rchscfa.2022.04.031 

Scientific articles

Search for baculoviruses in sawflies (Hymenoptera: Diprionidae) in Mexico

Cristian Estrada-Emigdio1 

Beatriz S. Macario-Tovar2 

Estefan Miranda‑Miranda2 

Raquel Cossio‑Bayugar2 

Ernesto González-Gaona3 

Karla V. De Lira-Ramos3 

Alejandro Pérez-Panduro1  * 

1Colegio de Postgraduados, Fitosanidad. km 36.5 carretera México-Texcoco. C. P. 56230. Texcoco, Estado de México, México.

2Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP). Centro Nacional de Investigaciones Disciplinarias en Salud Animal e Inocuidad (CENID-SAI). Búlevar Cuauhnahuac núm. 8534. C. P 62574. Jiutepec, Morelos, México.

3Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Campo Experimental Pabellón. Carretera Aguascalientes-Zacatecas, km 32.5. C. P. 20678. Pabellón de Arteaga, Aguascalientes, México.


Abstract

Introduction:

Sawflies (Hymenoptera: Diprionidae) affect conifers and other forest species in the northern hemisphere, reducing forest productivity and causing stand death, thereby justifying control actions.

Objective:

The aim was to collect baculoviral strains from Mexican sawfly populations and explore their potential for developing biopesticides.

Materials and methods:

A search for baculovirus-infected field populations was carried out. Twenty-three samples of live or dead larvae were collected from 12 localities. Microscopic diagnosis at 400X or 1000X under phase contrast, a DNA hybridization test, and a pathogenicity test were performed.

Results and discussion:

Samples from eleven localities showed baculoviral polyhedra. Three subsamples of Zadiprion ojedae DNA from Guachochi, Chihuahua, hybridized with a synthetic probe of the Neodiprion sertifer Polh gene sequence, confirming they harbored baculovirus infection.

Five tested inocula produced disease and mortality in all treated larvae; two of them induced baculoviral polyhedra proliferation in ≥89 % of the resulting cadavers. The rictus mortem of sawfly larvae killed by baculovirus differs from that observed in Lepidoptera: most baculovirus-bearing cadavers remain firm or hard, flattened or not, obscured or not, and do not hang from the hind legs. For most of the cadavers found with liquefied tissues, the agent that most probably caused death was not the baculovirus but the accompanying microbiota (bacilli, cocci, or fungi).

Conclusions:

Baculovirus strains are widely present in Mexican sawfly populations and several of them were pathogenic and promising for developing bioinsecticides.

Keywords: Diprionidae; Zadiprion; Neodiprion; Monoctenus; bioinsecticides

Resumen

Introducción:

Las moscas sierra (Hymenoptera: Diprionidae) afectan las coníferas y otras especies forestales en el hemisferio norte, reduciendo la productividad del bosque y causando la muerte de los rodales, lo que justifica acciones de control.

Objetivo:

Obtener cepas baculovíricas de poblaciones de moscas sierra mexicanas y explorar su potencial para elaborar bioplaguicidas.

Materiales y métodos:

Se hizo una búsqueda de poblaciones de campo infectadas con baculovirus. De 12 localidades se recolectaron 23 muestras de larvas vivas o muertas. Se realizó un diagnóstico microscópico a 400X o 1000X bajo contraste de fases, una prueba de hibridación de ADN y otra de patogenicidad.

Resultados y discusión:

Las muestras procedentes de once localidades mostraron poliedros baculovíricos. Tres submuestras de ADN de Zadiprion ojedae de Guachochi, Chihuahua, hibridaron con una sonda sintética de la secuencia del gen Polh de Neodiprion sertifer, confirmando que albergaban la infección baculovírica. Cinco inóculos probados produjeron enfermedad y mortalidad en todas las larvas tratadas; dos de ellos indujeron proliferación de poliedros baculovíricos en ≥89 % de los cadáveres resultantes. El rictus mortem de las larvas de moscas sierra muertas por baculovirus difiere del observado en los lepidópteros: la mayoría de los cadáveres portadores de baculovirus permanecen firmes o duros, aplanados o no, obscurecidos o no y no cuelgan de las patas traseras. La licuefacción de sus tejidos internos, su aplanamiento y obscurecimiento dependen de la abundancia relativa y tipo de microbiota acompañante. En la mayoría de los cadáveres encontrados con tejidos licuados, el agente que probablemente causó la muerte en mayor medida no fue el baculovirus, sino la microbiota acompañante (bacilos, cocos y hongos).

Conclusiones:

Las cepas baculovíricas están ampliamente presentes en las poblaciones mexicanas de moscas sierra y varias de ellas fueron patogénicas y prometedoras para el desarrollo de bioinsecticidas.

Palabras clave: Diprionidae; Zadiprion; Neodiprion; Monoctenus; bioinsecticidas.

Highlights:

  1. The first search for baculoviruses in sawflies is presented.

  2. Ten populations of eight sawfly species of Mexico yielded eleven samples baculoviral inocula.

  3. A hybridization test confirmed the presence of baculoviral DNA in our samples.

  4. Two of the five tested baculoviral inocula exhibited high potential as bioinsecticides.

  5. Rictus mortem of sawflies infected with the baculovirus differs from that of lepidopterans.

Introduction

Sawflies (Hymenoptera: Diprionidae) are phytophagous species that defoliate conifers of the genera Pinus, Juniperus, Cupressus, Abies and Larix, as well as some other broadleaf species in the northern hemisphere (Olivo, 2011; Smith, 1988, 1993). In Mexico, there have been significant outbreaks of sawflies in various forest areas; for example, in the Meseta Tarasca (Michoacán), between 1930-1943 and 1966-1975, there were diprionid infestations covering between 7 000 and 60 000 ha (González-Gaona et al., 2014), and in Chihuahua several infestations have been recorded since 1980 (Olivo, 2011) which have covered extensions of 3 400 to 34 500 ha (González-Gaona et al., 2014; Nolasco-Gumeta, 2014). These pests have also been detected in Guerrero, Oaxaca, and San Luis Potosí (Smith, Sánchez-Martínez, & Ordaz-Silva, 2010). This working group has also observed outbreaks of various sizes and intensities of these pests in Jalisco, Veracruz, and Sonora, but the record has not been documented in scientific publications. Other outbreaks have also been reported only in papers of limited circulation such as theses or symposia, a situation that prevents their citation in publications of greater impact. For a long time, it has been thought that only a few species of sawflies of the genera Zadiprion, Neodiprion, and Monoctenus are present in Mexico (Smith et al., 2010); however, the recent work by De Lira Ramos, González Gaona, and Sánchez Martínez (2021) has significantly increased the list with several newly described species and it will likely continue to grow as this group is further studied.

The damage caused by sawfly outbreaks is significant enough to justify control actions. Chemical insecticides are inadequate for such control because of their high potential to disrupt the environment. For this purpose, entomopathogens are preferable to insecticides for pest control, but even among entomopathogens there are groups that have significant disadvantages for forested areas, making some less ideal than others. For example, because of their wide host range, fungi such as Beauveria and Metarhizium are potentially harmful to non-target insects. At the same time, strains of the bacterium Bacillus thuringiensis Berliner, with potential against diprionids, also pose a risk to forest-dwelling bees and ants. In contrast, baculoviruses tend to have a high degree of specificity and aggressiveness towards their hosts, as well as mechanisms to persist in the environment (Balla et al., 2021; Thompson, Scott, & Wickman, 1981) and are therefore more suitable as control agents in forest environments.

In Mexico, no baculovirus inocula have been isolated from diprionids and no baculovirus-based bioinsecticides have been developed (Tapia-Uriza et al., 2022) against these pests, but several facts have prompted this search. Information exists documenting the occurrence of baculoviral infections in sawfly populations and the development of commercial bioinsecticides from wild inocula in several regions of the world (Arthurs & Dara, 2019; Dixon, 2019; Lucarotti, Morin, Graham, & Lapointe, 2007; Moreau et al., 2005; Moreau & Lucaritti, 2007; Qinghua et al., 2018; van Frankenhuyzen, Lucarotti, & Lavallée, 2016), suggesting that they could also exist in Mexico. Other studies note the existence of latent baculoviral infections in healthy lepidopteran populations (Hughes, Possee, & King, 1997; Il'inykh & Ul'yanova, 2005; Williams, Virto, Murillo, & Caballero, 2017), suggesting that sawflies could also harbor such infections. The experience of the members of this research team, who have found and activated latent infections in Lepidoptera, suggests, as a working hypothesis that these viruses could also be found in Diprionidae species. In this sense, the objective of the study was to search for baculoviral infections in Mexican populations of sawflies and to explore the potential of the obtained viral strains for the development of bioinsecticides.

Materials and methods

Sample collection and baculovirus detection

In this work, several populations of sawflies associated with Pinus and Juniperus trees that inhabit temperate forests were sampled. The samples involved populations located between 1 500 and 3 000 m in all mountain systems of Mexico (Figure 1). The location of the sawfly populations (pest outbreaks) was supported by the network of local technicians of CONAFOR (National Forestry Commission), and a number of them were detected in several forest regions. Subsequently, 23 samples of larvae suspected of carrying baculoviral infection were collected between 2011 and 2019. Most of the samples were conform by dead and dehydrated larvae, which were placed in containers either individually or in small groups of individuals with the same appearance. When the collected individuals were still hydrated, they were placed in vials with silica gel, or their confinements were placed on ice for transport to the laboratory. All samples were frozen until microscopic diagnosis. Two of the samples were collected as live larvae but they died during transport to the laboratory.

The samples were examined for the presence of baculovirus polyhedra in the cadavers. For diagnosis, a frotis from the abdominal region of each cadaver was prepared on a slide and observed under an Olympus CX43 microscope at 400X or 1000X with phase contrast or darkfield illumination; in addition to the appearance of each cadaver, the type of microorganism observed, and its relative abundance was also recorded. To identify the sawfly species involved in each field sample, adults obtained in the field or laboratory were processed with appropriate taxonomic keys (De Lira et al., 2021); however, for four samples (1, 2, 9, and 10, Table 1) no adults were collected or brooded from larvae. For other five samples 3, 7, 14, 15, and 23 (Table 1), only the genus was determined due to the lack of appropriate taxonomic keys. The adults derived from these samples did not consistently match the available taxonomic keys, suggesting that they may be new species.

Figure 1 Map showing the locations where the 23 samples of sawfly larvae were collected to search for baculoviral infections. The box on the right shows the list of samples analyzed and the name of the collection sites. 

Molecular detection of baculovirus DNA in larvae

A nucleic acid hybridization test was carried out on DNA from larval cadavers of Zadiprion ojedae Smith collected in Guachochi, Chihuahua, and Zadiprion borjai (González et al., 2022) from Miquihuana, Tamaulipas.

Probe preparation

The sequence of the polyhedrin (Polh) gene of Neodiprion abietis Harris nucleopolyhedrovirus (NeabNPV), with 741 bp, according to GenBank record NC_008252.1, was prepared by Integrated DNA Technologies (IDT). This sequence was integrated into the PuclDT plasmid and used to transform an Escherichia coli HB 101 competent cell line (Promega Corporation), following the manufacturer's instructions (Promega information bulletin TB092). Bacteria transformed with the PucIDT-NeabNPVPolh plasmid were incubated in tubes containing 50 mL of Luria-Bertani (LB) medium and 50 μg of ampicillin, at 37 °C for 18 h with constant shaking. The resulting bacterial culture was processed for plasmid DNA extraction with the miniprep procedure described by Green and Sambrook (2012). To generate the DIG-NeabNPVPVPolh probe used in this hybridization assay, 5 μg of the DNA obtained from the miniprep was used, labeled with hapten digoxigenin using the random primer method, following the instructions of the Dig-High prime DNA labeling kit (Roche).

DNA extraction from samples

From each field sample, groups of 10 specimens were frozen at -190 °C and ground in a porcelain mortar to a fine powder. From this powder, DNA was extracted using the phenol-chloroform procedure described by Green and Sambrook (2012). DNA from each group of powdered cadavers provided a sample for the DNA hybridization assay.

Hybridization assay

Two 100 ng aliquots of DNA from each group of larvae (Table 2) were placed on a nitrocellulose membrane (BioRad) in a Hoeffer PR600 Slot-Blot. Each aliquot was placed in a different slot, so each sample in the assay was processed twice. The pore size of the nitrocellulose membrane was 0.45 μm. DNA was attached to the membrane by vacuum. Subsequently, the DIG-NeabNPVPolh probe was applied following the procedure described by Green and Sambrook (2012). After the hybridization period, the membranes were washed and treated with alkaline phosphatase pre-labeled monoclonal antibodies and revealing reagents included in the Dig-High prime DNA labeling kit, following the product instructions. When necessary, the concentration of DNA in the working solutions was quantified using a NanoDrop® by reading the absorbance of each aliquot at 260 nm.

Biological activity assays of baculoviral inocula

One cadaver from each of five samples (4, 7, 8, 14, and 16; Table 1) was used as inoculum source to treat a group of healthy larvae. Inocula were prepared by macerating the donor cadaver with a micropistil in 1.5 mL Eppendorf tubes with distilled, sterilized water (1.0 mL) and 0.05 % Tween 80. The inoculum obtained from each of the first four donor cadavers was applied on larvae (19 to 22) of Zadiprion spp. from Xicotes, Atzalán, Veracruz, while the fifth inoculum was used on 145 larvae of Monoctenus cuauhtemoci (De Lira et al., 2022) from Ixcateopan de Cuauhtémoc, Guerrero (Table 3). A control group of 20 healthy larvae was used for inocula 4, 7, 8 and 14, while another of 100 healthy larvae was used for inoculum 16. In the latter case, the field inoculum was increased by several healthy larvae from the same locality, one year earlier. The abundance of baculovirus polyhedra was different in each donor cadaver.

The larvae treated (both Zadiprion spp. and M. cuauhtemoci) were inoculated by impregnating baits made of their natural died (pine needles or branchlets of Juniperus flacida Schltdl., respectively. The baits were impregnated by immersion of pieces of pine needles or branchlets of J. flacida (about 6 cm long) in the macerate, with the respective inoculum. The control groups were immersed in distilled water. The baits were stuck in floral sponge (cubes of approximately 2 x 2 x 2 cm) saturated with distilled water to keep them alive, turgid and erect, while the larvae fed on them. The baits were then placed inside cylindrical transparent plastic enclosures of approximately 10 x 10 cm (diameter x height). These containers were provided with ventilation holes of approximately 1.0 cm in diameter, covered with fine mesh fabric. When the larvae consumed the baits impregnated with inoculum, fresh food (not impregnated with inoculum) was provided. Containers were placed in an insectary with 12:12 h (light: dark), temperature of 25 °C, and relative humidity of 70 %. Every day the treated larvae were checked to collect those that had died, preserve them in containers by dehydration with silica gel, and then freeze them.

Results

Sample collection

In the field, several sawfly outbreaks were detected in 10 Mexican regions located into 8 Mexican states (Aguascalientes, Chihuahua, Durango, Guerrero, Jalisco, Michoacán, San Luis Potosí and Tamaulipas). From those outbreaks, 23 samples of larvae suspected of being infected by baculovirus were collected (Table 1). These samples included eight species of sawflies (Zadiprio falsus Smith, Z. ojedae, Z. borjai, Zadiprion spp., M. sanchezii Smith, M. cuauhtemoci, Neodiprion omosus Smith, N. autumnalis Smith, and Neodiprion spp.). Other species may be present in the four samples where neither genus nor species was determined because no adults were obtained from the corresponding population, or in the other five samples whose adults were not in accordance with the descriptions of the taxonomic keys, probably because they are new species.

Table 1 Microscopic diagnosis (400X and 1000X with phase contrast or darkfield) of cadaver samples of Diprionidae larvae collected in regions of Mexico. 

Sample Sample identification and number of individuals examined (n)* Comments and collection date Result
1 Property La Barranca, Armadillo de los Infantes, San Luis Potosí (10) 2013 10 with cocci and microsporidia
2 Los Alamitos, San José de Gracia, Sierra Fría Aguascalientes (10) 2013 10 with cocci
3 Zadiprion spp. (10) Pueblo Nuevo, Durango. 2013 Black cadaver preserved in group and partially disintegrated 10 with bacilli, microsporidia and cocci
4 Zadiprion falsus (10) El Pochón, Pueblo Nuevo, Durango. 2013 1 with abundant polyhedra and a few cocci. Liquefied body. 9 with bacilli, microsporidia and cocci
5 Zadiprion falsus (10) Pueblo Nuevo, Durango. 2013 Yellowish brown cadavers Preserved in groups and partially disintegrated. 10 with bacilli, microsporidia and cocci
6 Zadiprion falsus (10) Cruz de Piedra, Pueblo Nuevo, Durango. 2013 10 with bacilli, microsporidia and cocci
7 Zadiprion spp. (10) "Virus" Zerahuaran, Guachochi, Chihuahua. 2013 liquefied bodies 10 with fungal hyphae, very few polyhedra
8 Zadiprion falsus (10) "Virus" jaula 16, Pochón, Pueblo Nuevo, Durango. 2013 liquefied bodies 10 with small bacilli, few polyhedra
9 Guachochi, Chihuahua (10) 2013 10 with bacilli and cocci
10 San Juan Parangaricutiro, Michoacán (10) 2013 10 with cocci
11 Zadiprion falsus (10) San Juan Potreros Chimaltitlán, Jalisco. 2013 10 with cocci and microsporidia
12 Zadiprion falsus (10) Cruz del muchacho, Gómez Farías, Jalisco. 2012 10 with bacilli and microsporidia
13 Zadiprion falsus (10) Cruz del muchacho, Gómez Farías, Jalisco. 2012 Black dehydrated conserved in gel caps 10 with bacilli and microsporidia
14 Zadiprion spp. (41) Unión de San Isidro de Montes de Oca, Sierra de Ixtapa, Guerrero. 2019 Collected from needles on low branches. Black, flat and hard or leathery bodies. 1 with highly abundant polyhedra 3 with few polyhedra, long bacillus, and microsporidia. 37 with scarce polyhedra, small ovoid bacillus
15 Zadiprion spp. (187) Unión de San Isidro de Montes de Oca, Sierra de Ixtapa, Guerrero. 2019 Dry hard cadavers collected at the foot of the trees. 85 incubated in moist chamber. 60 Incubated in PDA. 42 cadavers examined under optical microscope. 145 with contaminating fungi. Medium bacillus and a Beauveria bassiana strain. 38 with abundant polyhedra, 4 cocci and bacilli
16 Monoctenus cuauhtemoci (25) Ixcateopan, Guerrero. 28/05/19 Hard cadavers. Larvae transported by commercial service and died during transport 1 with polyhedra 6 with abundant cocci 10 with few cocci. 8 with cocci, microsporidia, and bacilli
17 Monoctenus cuauhtemoci (75) Ixcateopan, Guerrero. 28/05/19 Hard cadavers Collected live but died during transport. 3 with scarce polyhedra 72 with cocci
18 Zadiprion ojedae (30) Guachochi, Chihuahua. 2019 Hard, flat cadavers 8 with polyhedra and cocci 22 with cocci and/or hyphae
19 Zadiprion borjai (21) Miquihuala, Tamaulipas. 13/12/2017 Died during transport from Tamaulipas to Texcoco. Hard, flat bodies 13 with polyhedra 8 with cocci
20 Monoctenus sanchezii (5) Armadillo de los Infante, San Luis Potosí. 17/09/2011 Firm, but soft, cylindrical, brownish yellow cadavers 1 with abundant polyhedra 1 with very few polyhedra 3 with cocci
21 Neodiprion omosus (5) Los Alamitos, San José, Sierra Fría, Aguascalientes. 08/08/2011 Flat, dry, hard, dark cadavers 5 with small globose bacilli
22 Neodiprion autumnalis (9) Guachochi, Chihuahua. 29/05/2012 Firm but soft cylindrical cadavers, brownish yellow in color. 3 with abundat polyhedra 2 with very few polyhedra and cocci 4 with cocci
23 Neodiprion spp. (8) San Juan Pueblo Nuevo, Parangaricutiro, Michoacán. September 2011 Hard, dry, dark brown cadavers. 8 with small globular bacilli

* When the species is not indicated is due to the lack of adults in the sample or adequate taxonomic keys.

Molecular detection

The DIG-NeabNPVPolh probe hybridized with its homologous sequence from the positive control (NeabNPVPolh sequence) placed in wells 3A and 3B in Figure 2 but did not produce significant signal in the two negative controls: tick DNA (1A and 1B) and DNA from healthy Zadiprion spp. cell culture (wells 2A and 2B). The test showed significant reaction consistent with DNA from three Z. ojedae subsamples from Guachochi, Chihuahua (wells 4A and 4B; 8A and 8B; and 12A and 12B in Figure 2). The remaining samples did not show consistent responses between replications; therefore, they were considered negative.

Table 2 DNA samples from Zadiprion ojedae and Z. borjai cadavers subjected to hybridization tests with the DIG-NeabNPVPolh probe. 100 ng of each DNA sample was used in each well. All DNA samples were processed in duplicate. 

Sample and replication Sample description and collecting date
1 A DNA from tick (Rhipicephalus microplus)
B
2 A DNA from healthy Zadiprion sp. cells
B
3 A PucIDT-Polh NeabNPV
B
4 A Zadiprion ojedae. Guachi, Chihuahua (140719)
B
5 A Zadiprion borjai. Ejido Servando Canales, Miquihuana, Tamaulipas (090719)
B
6 A Zadiprion ojedae. Guachochi, Chihuahua (060619)
B
7 A Zadiprion ojedae. Guachochi, Chihuahua (060619)
B
8 A Zadiprion ojedae. Guachochi, Chihuahua (100619)
B
9 A Zadiprion borjai. Ejido Servando Canales, Miquihuana, Tamaulipas (090719)
B
10 A Zadiprion ojedae. Guachochi, Chihuahua (100619)
B
11 A Zadiprion ojedae. Guachochi, Chihuahua (190719)
B
12 A Zadiprion ojedae. Guachochi, Chihuahua (190719)
B

Figure 2 Hybridization assay of DNA from Diprionidae larval cadavers on a nitrocellulose membrane. The sequence of the NeabNPV Polh gene (741 bp), replicated in the PucIDT plasmid and labeled with digoxigenin, was used as a probe. 100 ng of DNA was used in each well. Each sample was formed from 10 cadavers of Zadiprion ojedae and Z. borjai larvae collected in the field and suspected of carrying baculoviral infection. Hybridization was detected by enzyme-linked immunosorbent reaction using a chromogenic substrate that produces a dark staining. 

Detection of baculoviral infection by light microscopy

Small shiny bodies were found in frotises of larval abdominal tissue observed under the microscope (Figure 3); their brightness, shape and abundance made them compatible with baculoviral polyhedra and different from other shiny objects also present in the frotises. These baculoviral polyhedra were detected in cadavers from 11 field samples (4, 7, 8, 14, 15, 16, 17, 18, 19, 20 and 22) taken from five Mexican states: Chihuahua, Durango, Guerrero, San Luis Potosí and Tamaulipas (Table 1). The cadavers with polyhedra also presented other microorganisms such as bacteria (cocci or bacilli) (Figure 3), microsporidia, or fungi. Frequently, two or more of these groups of microorganisms were found in each cadaver concomitant with polyhedra.

Figure 3 Macrophotographic section taken from a frotis of the abdominal region of a cadaver of Monoctenus cuauhtemoci. from Ixcateopan de Cuauhtémoc, Guerrero (sample 16 in Table 1). Phase contrast illumination and 400X magnification were used for this image. The circles surround small shiny bodies interpreted as baculiviral polyhedra. Arrows point to bacillary bacterial cells with a shiny body at each end, suggesting that they may belong to some variant of Bacillus thuringienesis. Rectangles surround cocci-like bacteria. 

Biological activity of cadaveric inocula

The cadaveric inocula explored induced symptoms and signs of disease compatible with baculoviral or bacterial infections: test larvae stopped feeding, gradually lost motility and died with generalized darkening of the body or localized in their abdominal area. In contrast, about 85 % of the larvae in the control groups reached the pupal stage or remained healthy until the last treated larva died. Microscopic examination of most of the cadavers resulting from larvae treated with inocula 4 and 14 showed abundant polyhedra (in 17 of 19 and 22 of 22, respectively). According to Table 3, inocula 7 and 8 induced sparse polyhedra in few cadavers (in 1 of 20 and in 6 of 20, respectively). Inoculum 16 induced a moderate frequency of polyhedra in the resulting cadavers (33 out of 145). All macerates induced, in addition to polyhedra, proliferation of other microorganisms distinguishable under light microscopy at 400X or 1000X and phase contrast or darkfield illumination. Such microorganisms were bacilli (small, medium or large), cocci, microsporidia or fungal hyphae. All of these constituted the accompanying microbiota of the polyhedra in the field samples and in the cadavers resulting from this assay, part of which is shown in Figure 3. The accompanying microbiota in most of the larvae treated with inocula 4 and 14 were bacilli. Thirteen of the 20 larvae treated with inoculum 7 showed proliferation of fungal hyphae similar to those observed in the inoculum donor cadaver and bacteria (Table 3). Three other cadavers from larvae treated with inoculum 7 or 8 showed cocci-like bacteria (Table 3). Most of the larvae treated with inoculum 16 showed small to medium-sized bacilli or fungal hyphae.

Table 3 Inoculation of Zadiprion spp. and Monoctenus cuauhtemoci larvae with macerate from polyhedra-bearing cadavers. Most of the treated larval cadavers had more than one type of microorganism simultaneously. 

Inoculum source Characteristics of resulting cadavers Cadaver diagnosis (n/treated)
Inoculation of Zadiprion spp. de Xicotes larvae, Atzalán, Veracruz
Zadiprion falsus El Pochón, Pueblo Nuevo, Durango (Sample 4 from Table 1). Flat dark bodies Abundant polyhedra (17/19) Bacilli: small (10/19) and medium (8/19).
Zadiprion spp. "Virus" Zerahuaran, Guachochi, Chihuahua (Sample 7 from Table 1). Zadiprion spp. Some with liquified bodies and others flat Very few polyhedra (1/20) Bacilli: small (6/20), medium (5/20) and large (8/20) Fungal hyphae (13/20), and cocci (2/20).
Zadiprion falsus "Virus" cage 16, El Pochón, Pueblo Nuevo, Durango (Sample 8 from Table 1). Bodies flat and dark, some liquefied Scarce polyhedra (6/20) Bacilli: small (20/20), medium (9/20) cocci (1/20).
Zadiprion spp. Hard cadaver. Ixtapa, Guerrero (Sample 14 from Table 1). Flat bodies Polyhedra (22/22) Bacilli: small (2/22), medium (6/22), large (10/22).
Inoculation of Monoctenus cuautemoci larvae from Ixcateopan de Cuauhtemoc, Guerrero
Monoctenus cuauhtemoci Ixcateopan de Cuauhtémoc, Guerrero. Hard cadaver (Sample 16 from Table 1). Flat bodies. Inoculum incremented in M. cuauhtemoci from Ixcateopan, Guerrero. Polyhedra (33/145) Small bacilli (54/145) Medium bacilli (22/145) Hyphae (18/145)

Discussion

Positive evidence in favor of the working hypothesis was obtained: baculoviral infections were detected in 11 of the 23 sawfly samples explored; inocula were obtained from the infected samples; five inocula were tested, of which two showed great potential for developing bioinsecticides based on baculoviruses. In this study, the appearance of sawfly larvae affected by baculovirus differed from that observed in lepidopteran larvae. Other pathogenic microorganisms such as bacilli, cocci, microsporidia, and fungi were also present in the cadavers together with the viruses.

Detection of baculoviral infections

The microscopic diagnosis of larval cadaveric frotises at 400X and 1000X indicated the presence of small shiny bodies in 11 of the 23 field samples, which were interpreted as baculoviral polyhedra based on their brightness, shape, and relative abundance (Table 1). The samples harboring baculoviral infections comes from 6 of the 11-collection region explored, into five Mexican states. Eight sawfly species were represented in the samples harboring baculoviral polyhedra: Z. falsus, Z. ojedae, Zadiprion borjai, Zadiprion spp., M. sanchezii, M. cuauhtemoci, N. omosus, and N. autumnalis (Table 1), which are distributed in three genera.

The interpretation of those shiny bodies as baculoviral polyhedra is supported by two pieces of evidence provided in this study. 1) Successful induction of disease in healthy larvae inoculated with any of five macerates of cadavers harboring polyhedra and the subsequent proliferation of polyhedra in the cadavers resulting from such inoculations (Table 3). 2) Hybridization of three DNA subsamples from the Z. ojedae population located in Guachochi, Chihuahua (locus 4, 8, and 12 in Figure 2) with the DIG-NeabNPVPolh probe, indicating the presence of baculoviral DNA in the samples. Other larvae from Guachochis’s Z. ojedae population showed polyhedra on microscopic examination (sample 18, Table 1). In agreement with this evidence, the small shiny bodies observed in cadavers of the other 10 samples (Table 1) not used in this hybridization test were also interpreted as baculoviral polyhedra: 4 and 8 (Zadiprion falsus); 7, 14, 15 (Zadiprion spp.); 19 (Z. borjai); 16 and 17 (M. cuauhtemoci); 20 (M. sanchezii); and 22 (N. autumnalis).

The DIG-NeabNPVPolh probe is considered highly specific for detecting the sawfly nucleopolyhedrovirus polyhedrin gene, as its sequence, the same as that of the NeabNPV Polh gene, only differs by 5 % from the homologous sequence of any of the other known nucleopolyhedroviruses in Diprionidae; for example, those of N. lecontei (NeleNPV) and N. sertifer (NeseNPV) (Lauzon et al., 2006). That maximum expected divergence between the probe used here and its target DNA does not represent an impediment to hybridization, as suggested by the successful use of other probes in detecting their respective DNA targets with divergences >20 % (Meier-Kolthoff et al., 2014).

Cadaver appearance (rictus mortem)

In the literature, the only description found of baculovirus-induced rictus mortem in these insects indicates that larvae hang from their hind legs (Dixon, 2019). Based on that description and this working group's experience with baculoviral infections in Lepidoptera, we searched for sawfly larval cadavers with that appearance in field. They were expected to be dark, and flattened with liquefied internal tissues, as described for lepidopteran larvae killed by baculoviruses (Federici, 1997). Although larvae with liquefied internal tissues were observed in three of the first samples with polyhedra, subsequent samples showed that this appearance is the least frequent in baculovirus-killed diprionids. Two samples from the same population of Zadiprion spp. (14 and 15; Table 1 and Figure 4) clearly show that the most common consistency of Diprionidae cadavers with polyhedra differs from that commonly observed in Lepidoptera (hanging from their hind legs, liquefied, and flattened).

The first of these samples, number 14 (Table 1; Figures 4A and 4B), consisted of cadavers found on needles of low branches of pine trees. They were either hanging by their hind legs or fell from the highest branches; their bodies were flattened, black in color and had a leathery or hard consistency. Only one of them showed abundant polyhedra, while the other 40 showed few or scarce polyhedra. About 97 % of the cadavers in this sample presented bacilli as the dominant microorganism, so their death was attributed to bacillus and not to virus, despite the presence of the latter in the cadavers.

Figure 4 Appearance of sawfly (Zadiprion spp.) larval cadavers in the field (La Unión de San Isidro Montes de Oca, Sierra de Ixtapa, Guerrero), which carried baculoviral polyhedra. A and B are typical in sample 14; C, D, and E are typical in sample 15 in Table 1. The formers are fully or partially obscured. The latters are only partially obscured, depending on the relative abundance of the accompanying microbiota in the cadavers. 

Cadavers in sample 15 (Table 1; Figures 4C, 4D and 4E) were found on the ground at the foot of pine trees. It is assumed that they climbed down the trunk before dying, possibly to pupate. The cadavers were found dry and hard (not liquefied), retaining their cylindrical shape and, most of them, even their natural color (yellowish), while others showed black areas on their body. Because these characteristics suggest a fungal death, the first 145 cadavers examined from that sample were placed in a humid chamber or small pieces of them were directly deposited on PDA culture media. Subsequently, for the next 42 cadavers, frotises were prepared from their abdominal region; under light microscopy it was observed that polyhedra dominated over concurrent microbiota in more than 90 % of them (38 of 42), and only four cadavers had cocci and bacilli as dominant microbiota.

When comparing the appearance of Zadiprion spp. cadavers in samples 14 and 15 and their accompanying microbiota, it was clear that the flat body and dark color observed in sample 14 were related to the bacilli and not to the virus. Similarly, the hard, cylindrical body of the cadavers in sample 15 was associated with the virus and not with the accompanying microbiota. Another piece of evidence suggesting that baculovirus-killed sawfly larvae remain hard or leathery, but not liquefied, comes from Lucarotti et al. (2007), who describe how they preserve and process large quantities of baculovirus-killed N. abietis larvae for the formulation of a bioinsecticide.

The liquefied appearance of the cadavers was observed in one individual from sample 4, in 10 from sample 7 and in 10 from sample 8. The liquefied cadaver from sample 4 showed few cocci and many bacteria and polyhedra; while those from samples 7 and 8 showed few polyhedra, but a lot of accompanying microbiota (Table 1). Subsequently, when the macerate of a cadaver from sample 4 was inoculated into healthy larvae, it induced a high frequency of cadavers with polyhedra (17 out of 19) and a large amount of polyhedra in each cadaver; while a macerate from sample 7 induced only one cadaver with very few polyhedra, but abundant accompanying microbiota in all treated larvae (Table 3). Therefore, in cadavers carrying polyhedra, characteristics such as liquefaction, flattening and darkening of the body, and hanging from the hind legs, appear to be associated with the presence, type, and relative abundance of the accompanying microbiota, but not with baculovirus.

When the baculovirus was the predominant death agent in the sawfly cadavers, most cadavers conserved its cylindrical shape, their bodies were hard and did not hang from their hind legs. This rictus mortem is different from that induced by the baculovirus in lepidopteran species: which get liquefied, flatted, torn, and commonly, hang from their hind legs (Federici, 1997). The differences most likely arise from the fact that, in diprionids, only mesenteron cells are attacked (Harrison et al., 2018), leaving the rest of the tissues intact (unless degraded by other microorganisms); while in Lepidoptera, almost all tissues are colonized by the virus, which became liquefied by (Federici, 1997). This description of the rictus mortem of sawfly larvae killed by baculoviruses may be useful in future searches for these viruses for controlling forest pests.

Bioinsecticidal potential of baculoviruses

The five inocula tested showed differential potential as bioinsecticides. Although all of them produced death in all treated larvae, the identification of the agent causing death provides a first appreciation of the relative potential of each inoculum as a bioinsecticide. Inocula from samples 4 and 14 caused numerous polyhedra to appear on each cadaver and a high proportion of cadavers with polyhedra (17 of 19 and 22 of 22, respectively; Table 3). Samples 7 and 8 caused a small number of polyhedra in each resulting cadaver and only a few cadavers with polyhedra (1 of 20 and 6 of 20, respectively; Table 3).

Inoculum from sample 16 caused polyhedra proliferation in a moderate frequency of cadavers (33 of 145; Table 3). It was assumed that dead larvae that did not show polyhedra were killed by the microbiota that accompanied the virus in the donor cadavers. The ability of, at least one part of the microbiota accompanying the baculovirus to cause death is discussed below. No species of that microbiota was identified, but it was evident that the fungal hyphae observed on the cadavers in this trial do not belong to Metarhizum or Beauveria genera.

The abundance of polyhedra in the inoculum donor cadavers of samples 4 and 14, on the one hand, and 7 and 8, on the other, maintained a clear correspondence with the abundance of the polyhedra in the cadavers resulting from this test and with the frequency of cadavers bearing polyhedra (Table 3). This correspondence suggests that inocula from samples 4 and 14 have a high potential to be developed as bioinsecticides due to their high infectivity.

The inoculum of sample 14 provided also evidence of having high potential as a natural biocontrol factor in the field where it was collected (La Unión de San Isidro Montes de Oca, Sierra de Ixtapa, Guerrero). Since sample 15 (Table 1) (not included in this test) was taken from the same group of pine trees as sample 14, it is reasonable to assume that both samples harbor the same virus strain and that pathogens found in them performed together at the scene of good control of their host observed in the field. It was, the sawfly population was virtually eliminated by pathogens prior to sampling, and that happened very early in the development of this pest outbreak as suggested both by the appearance of the pupal cocoons found scratching in the ground, and by the evidently reduced extension of the infestation. Only two generations of the pest would have occurred in that place by the sampling time. So early elimination of the outbreak clearly suggests that this inoculum is highly infective on its host.

Both the owner of the field where the sample was collected and the CONAFOR technician (the official in charge of supervising forest health in that area) assured that there was no human intervention in the development of this epizootic. Although several pathogens were detected acting simultaneously (virus, bacteria, and at least one fungus), the virus turned out to be the most frequent in the cadavers collected there.

Regarding the inocula of samples 7, 8, and 16, in the first perception, this test suggests that they have a relatively low potential as bioinsecticides, because they produced a low frequency of cadavers with polyhedra. However, after a detailed look at the result, the expectation of good potential of these inocula as bioinsecticides is maintained for the following reasons: 1) the low frequency of cadavers with polyhedra may be due to the reduced number of polyhedra in donor cadavers; 2) larvae that received inocula 7 or 8 and, most of those receiving inoculum 16, showed abundant accompanying microbiota, but only a few showed some polyhedra suggesting pathogenic capacity in the microbiota; therefore, 3) inevitably, it competed with the virus and could displace it, either due to being more competitive or more abundant.

Based on this reasoning, it seems necessary to carry out further tests to adequately measure the potential of all the inocula obtained, that of the pathogenic microbiota accompanying the viruses, and the interactions between them. Such interactions seem very interesting for the management of these pests because they may involve synergies that should be exploited to achieve greater control of the pest, or antagonisms that should be avoided. In such new trials, it would be desirable to test the viral inocula and the accompanying microbiota, properly purified.

Another reasonable precaution would be to thoroughly search for and evaluate all existing virus strains of each sawfly species. Such evaluation of strains should be done in the stands where their host species is found, and the strain that provides the best control in each location could be used as a means of control in such areas. Failure to do so would run the risk of using, in some areas, a strain with less control potential than that of the resident strain and, due to the flooding application of a less potent exotic strain, the resident strain with greater potential would be displaced. Thus, virus strains with superior potential could be lost due to not having been discovered in a timely manner.

Conclusions

The results of this study clearly suggest the following three conclusions: 1) there are abundant baculoviral infections in Mexican sawfly populations, as they were present in 11 of 23 field samples; 2) several of the inocula obtained are pathogenic enough to make them promising for developing bioinsecticides based on them; 3) an exhaustive search for the existing baculoviral strains in all sawfly species and a proper selection of the best against each pest species would prevent the risk of losing local strains with high control potential as a consequence of flooding its niche with a foreign competitively inferior strain.

Acknowledgments

We thank the funding from the CONACYT-CONAFOR Sectorial fund, project CONAFOR 2017 CO2 no. 291304. We are also grateful for the facilities provided by the H. Ayuntamiento de Ixcateopan de Cuauhtémoc, Guerrero, and the regidores of Ecology: Víctor Leyva Guerrero and María Dolores Bustamante Cirilo. We thank Dr. Miguel Ángel González González, Dr. Arturo Corrales Suastegui and M. C. Jorge Valdez Carrazco.

References

Arthurs, S., & Dara, S. K. (2019). Microbial biopesticides for invertebrate pests and their markets in the United States. Journal of Invertebrate Pathology, 165, 13‒21. doi: 10.1016/j.jip.2018.01.008 [ Links ]

Balla, A., Silini, A., Cherif-Silini, H., Chenari Bouket, A., Moser, W. K., Nowakowska, J. A.,. ..Belbahri, L. (2021). The threat of pests and pathogens and the potential for biological control in forest ecosystems. Forests, 12(11), 1579. doi: 10.3390/f12111579 [ Links ]

De Lira Ramos, K. V., González Gaona, E., & Sánchez Martínez, G. (2021). Características generales de las moscas sierra de las coníferas. In E. González G., & K. V. De Lira R. (Eds.), Moscas sierra: Taxonomía, fenología, distribución y manejo (pp. 35‒82). Aguascalientes, México: INIFAP-CIRNOC-Campo Experimental Pabellón. [ Links ]

De Lira-Ramos, K. V., González-Gaona, E., Rodríguez-Cruz, Y. E., Piza-Núñez, E. G., & Gómez-Núñez, J. C. (2022). Nueva especie de Monoctenus (Hymenoptera: Diprionidae) ataca Juniperus flaccida Schltdl. (Cupressaceae) en Guerrero, México. Revista Mexicana de Ciencias Forestales, 13(69), 73-94. https://doi.org/10.29298/rmcf.v13i69.1093 [ Links ]

Dixon, W. (2019). Pine sawflies, Neodiprion spp. (Insecta: Hymenoptera: Diprionidae). Retrieved from https://edis.ifas.ufl.edu/pdf%5CIN%5CIN59200.pdfLinks ]

Federici, B. A. (1997). Baculovirus pathogenesis. In L. K. Miller (Ed.), The Baculoviruses. The Viruses (pp. 33‒59). Springer, Boston, MA: Springer. doi: 10.1007/978-1-4899-1834-5_3 [ Links ]

González-Gaona, E., Bonilla, T. F., Quiñones, B. S., Sánchez, M. G., Tafoya, R. F., España, L. M. P.,... Robles, U. S. (2014). Guía para la identificación de moscas sierra de la familia Diprionidae presentes en el centro norte de México. Mexico: INIFAP. Retrieved from https://docplayer.es/46918897-Guia-guia-para-la-identificacion-la-identificacion-moscas-sierra-la-familia-diprionidae-de-presentes-moscas-en-el-centro-norte-sierra.htmlLinks ]

González-Gaona, E., Borja-Nava, H. E, Lira-Ramos, D., Karla, V., Rodríguez-Cruz, Y. E., & Arriola-Padilla, V. J. (2022). Nueva especie de mosca sierra del género Zadiprion Rohwer (Hymenoptera: Diprionidae) atacando a Pinus cembroides (Zucc.) en Tamaulipas, México. Revista Chapingo Serie Ciencias Forestales, 28(3), 399‒409. doi: 10.5154/r.rchscfa.2021.10.061 [ Links ]

Green, M. R., & Sambrook, J. (2012). Molecular cloning. A laboratory manual (4th ed). USA: Cold Spring Harbor Laboratory Press. [ Links ]

Harrison, R. L., Herniou, E. A., Jehle, J. A., Theilmann, D. A., Burand, J. P., Becnel, J. J., Krell , …Bauchan, G. R. (2018). ICTV virus taxonomy profile: Baculoviridae. Journal of General Virology, 99(9), 1185‒1186. Retrieved from https://www.microbiologyresearch.org/content/journal/jgv/10.1099/jgv.0.001107?crawler=trueLinks ]

Hughes, D. S., Possee, R. D., & King, L. A. (1997). Evidence for the presence of a low-level, persistent baculovirus infection of Mamestra brassicae insects. Journal of General Virology, 78(7), 1801‒1805. doi: 10.1099/0022-1317-78-7-1801 [ Links ]

Il'inykh, A. V., & Ul'yanova, E. G. (2005). Latency of baculoviruses. Biology Bulletin, 32(5), 496‒502. doi: 10.1007/s10525-005-0131-1 [ Links ]

Lauzon, H. A., Garcia-Maruniak, A., Paolo, M. D. A., Clemente, J. C., Herniou, E. A., Lucarotti, C. J.,... Maruniak, J. E. (2006). Genomic comparison of Neodiprion sertifer and Neodiprion lecontei nucleopolyhedroviruses and identification of potential hymenopteran baculovirus-specific open reading frames. Journal of General Virology, 87(6), 1477‒1489. doi: 10.1099/vir.0.81727-0 [ Links ]

Lucarotti, C. J., Morin, B., Graham, R. I., & Lapointe, R. (2007). Production, application, and field performance of Abietiv™, the balsam fir sawfly nucleopolyhedrovirus. Virologica sinica, 22(2), 163. doi: 10.1007/s12250-007-0018-z [ Links ]

Meier-Kolthoff, J. P., Hahnke, R. L., Petersen, J., Scheuner, C., Michael, V., Fiebig, A.,... Klenk, H. P. (2014). Complete genome sequence of DSM 30083 T, the type strain (U5/41 T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy. Standards in Genomic Sciences, 9(1), 1‒19. doi: 10.1186/1944-3277-9-2 [ Links ]

Moreau, G., & Lucarotti, C. J. (2007). A brief review of the past use of baculoviruses for the management of eruptive forest defoliators and recent developments on a sawfly virus in Canada. The Forestry Chronicle, 83(1), 105‒112. doi: 10.5558/tfc83105-1 [ Links ]

Moreau, G., Lucarotti, C. J., Kettela, E. G., Thurston, G. S., Holmes, S., Weaver, C., …Morin, B. (2005). Aerial application of nucleopolyhedrovirus induce decline in increasing and peaking populations of Neodiprion abietis. Biological Control, 33(1), 6573. doi: 10.1016/j.biocontrol.2005.01.008 [ Links ]

Nolasco-Gumeta, A. (2014). Defoliadores de coníferas de los géneros Zadiprion spp. y Neodiprion spp. existentes en México. Tesis profesional. Departamento Forestal, División de Agronomía, UAAAN. Saltillo, Coahuila, México. Retrieved from http://repositorio.uaaan.mx:8080/xmlui/handle/123456789/3887Links ]

Olivo, M. J. A. (2011). Brotes epidémicos de diprionidos en la sierra Tarahumara de Chihuahua. M. A. Equihua, & V. Estrada E., J. A. Acuna S., & M. P. Cháires G. (Eds.), XV Simposio Nacional de Parasitología Forestal. Oaxaca, México: CONAFOR. Retrieved from https://www.uaeh.edu.mx/investigacion/icap/LI_IntGenAmb/Juana_Fons/8.pdfLinks ]

Qinghua Wang, Enjie Li, Na Li, Yuzhu Wang, Zhilin Zhang, & Yongan Zhang. (2018). Infection of a nucleopolyhedrovirus to Neodiprion zhejiangenis Zhou & Xiao (Hymenoptera: Diprionidae). Biocontrol Science and Technology, 28(8), 761‒771. doi: 10.1080/09583157.2018.1493089 [ Links ]

Smith, D. R. (1988). A synopsis of the sawflies (Hymenoptera: Symphyta) of America south of the United States: introduction, Xyelidae, Pamphiliidae, Cimbicidae, Diprionidae, Xiphydriidae, Siricidae, Orussidae, Cephidae. Systematic Entomology, 13(2), 205-261. doi: 10.1111/j.1365-3113.1988.tb00242.x [ Links ]

Smith, D. R. (1993). Systematics, life history and distribution of sawflies. In M. R. Wagner, & K. F. Raffa (Eds.), Sawfly life history adaptations to wood plants (pp. 3‒ 32). California, USA: Academic Press. Retrieved from https://www.researchgate.net/publication/260798522_Systematics_life_history_and_distribut9ion_of_sawflies#fullTextFileContentLinks ]

Smith, R. D., Sánchez-Martínez, G., & Ordaz-Silva, S. (2010). A new Monoctenus (Hymenoptera: Diprionidae) damaging Juniperus flaccida (Cupressaceae) in San Luis Potosí, México. Proceedings of the Entomological Society of Washington, 112(3), 444-450. doi: 10.4289/0013-8797.112.3.444 [ Links ]

Tapia-Uriza, T. R., Cossío-Bayúgar, R., González-Gaona, E., Lira-Ramos, K. V. D., Rodríguez-Cruz, Y. E., & Miranda-Miranda, E. (2022). Establecimiento de novo de un cultivo in vitro de una línea celular derivada del intestino de moscas sierra (Hymenoptera: Diprionidae). Revista Mexicana de Ciencias Forestales, 13(69), 95‒111. doi: 10.29298/rmcf.v13i69.1088 [ Links ]

Thompson, C. G., Scott, D. W., & Wickman, B. E. (1981). Long-term persistence of the nuclear polyhedrosis virus of the Douglas-fir tussock moth, Orgyia pseudotsugata (Lepidoptera: Lymantriidae), in forest soil. Environmental Entomology, 10(2), 254‒255. doi: 10.1093/ee/10.2.254 [ Links ]

van Frankenhuyzen, K., Lucarotti, C., & Lavallée, R. (2016). Canadian contributions to forest insect pathology and to the use of pathogens in forest pest management. The Canadian Entomologist, 148(S1), S210‒S238. doi: 10.4039/tce.2015.20 [ Links ]

Williams, T., Virto, C., Murillo, R., & Caballero, P. (2017). Covert infection of insects by baculoviruses. Frontiers in Microbiology, 8, 1337. doi: 10.3389/fmicb.2017.01337 [ Links ]

Received: April 26, 2022; Accepted: December 28, 2022

*Corresponding author: aperez@colpos.mx; tel.: +52 595 101 2254.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License