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Highlights:
Zadiprion llanderalae is described as a new species.
Pinus oaxacana, Pinus patula and Pinus leiophylla are attacked by a defoliator.
In the open field, an average density of 39.2 ± 9.69 sawfly cocoons∙m-2 was recorded.
The sawfly cocoons were attacked by wasps (parasitoids) of the genus Exenterus
Introduction
Forest fires, excessive logging, and pests and diseases are some of the problems affecting Mexico's forests (Salinas et al., 2010; Sosa et al., 2018). The most prominent pests include pine bark beetles and defoliators of the genera Zadiprion, Neodiprion and Monoctenus (Castro, 1981; Cibrián-Tovar et al., 1995; Smith et al., 2010).
Since the beginning of the 20th century, there have been records of the damage caused by the larvae of species of the genus Zadiprion in the forests of Mexico, whose adults are commonly called sawflies because they resemble flies of the order Diptera (Cibrián et al., 1995; Smith,1988). The species currently recognized in Mexico include Zadiprion townsendi (Cockerell) 1898, Zadiprion rohweri (Middleton) 1931, Zadiprion falsusSmith 1988, Zadiprion roteus Smith 1975, Zadiprion howdeni Smith 1975, Zadiprion ojedae Smith and Sánchez-Martínez 2012, Zadiprion jeffreyi Smith 2019 (Smith, 1993, 2019; Smith et al., 2012) and Zadiprion borjai González et al. 2021 (González-Gaona et al., 2022).
Severe infestations by a sawfly of the genus Zadiprion were reported in the Sierra Norte of Oaxaca from 2015 to 2021. This species was initially identified as Z. falsus (Suárez-Mota et al., 2018) and subsequently as Z. howdeni (Aguirre-Hidalgo et al., 2020). The present study, after a detailed review of field-collected and laboratory-reared specimens, showed that the external morphological and ovipositor characteristics of the female do not correspond to Z. falsus, Z. howdeni or any of the other species so far recognized. Consequently, it was concluded that this is an undescribed species, which is reported for the first time in this research.
The municipalities affected by the species studied are Ixtlán de Juárez, Santa Catarina Ixtepeji, Santiago Xiacui, Santa María Jaltianguis, Nuevo Zoquiapam and Pueblos Mancomunados (Unión Estatal de Silvicultores Comunitarios del Estado de Oaxaca [UESCO], 2018). Infestations were recorded from 2015 to 2021, with 12 194 ha being affected in the latter year. Therefore, control actions were developed during 2019, 2020 and 2021, mainly applying biological insecticides sprayed by helicopter (Comisión Nacional Forestal [CONAFOR], 2021). The species Pinus oaxacana Mirov., Pinus patula Schiede ex Schltdl. & Cham., Pinus pseudostrobus Lindl. and Pinus teocote Schiede ex Schltdl. & Cham. have been recorded as main hosts, with P. oaxacana as the preferred host (Aguirre-Hidalgo et al., 2020; Suárez-Mota et al., 2018). These authors also indicate that, in the study area, Zadiprion adults are present from June to October, larvae from June to November, and new cocoons from September to December. However, the inhabitants of the communities collected larvae from several instars in December, January, and February, leading to a hypothesis that there are two generations per year or that several species of Zadiprion exist with cycles that are out of phase in date.
Although scientific literature and a CONAFOR report refer to Z. falsus as the pest species in the Sierra Norte de Oaxaca, González-Gaona and Sánchez-Martínez (2018) mention that the morphology and life cycle dates do not match this species; therefore, it was necessary to corroborate its identity. This motivated the present study in which the objectives were to describe the new species and gather information regarding its biology.
Materials and Methods
The study was carried out in forest areas of the municipality of Santa Catarina Ixtepeji, located at coordinates 17° 16´ N and 96° 34´ W (Figure 1), with an average altitude of 1 920 m, belonging to the district of Ixtlán de Juárez in the Sierra Madre Oriental, locally known as Sierra Juárez or Sierra Norte de Oaxaca (Instituto Nacional de Geografía e Informática [INEGI], 2016; Ortiz-Pérez et al., 2004).
The main vegetation types in this area are pine-oak forest and oak forest. The species P. teocote, P. oaxacana and Pinus leiophylla var. leiophylla Schiede ex Schltdl. & Cham are the most representative, while Quercus crassifolia Humb. & Bonpl., Quercus castanea Née, Quercus rugosa Née and Pseudotsuga menziesii var. oaxacana Debreczy & I. Rácz have a lower presence (Castellanos-Bolaños et al., 2010).
Figure 1 Geographical location of the study site in the municipality of Santa Catarina Ixtepeji, Oaxaca.
Egg collection and measurement
From April to July 2021, surveys were conducted for the detection of pine needles with recent ovipositions of the sawfly under study, including females that usually remain after oviposition to care for their eggs. Length and width of 78 eggs were measured using Leica Application Suite V 4.6.1® software and a Leica DFC295® camera mounted on a Leica M80® stereo microscope.
Larval collection and measurement
From August 24, 2020 to July 29, 2021, nine field trips were conducted at intervals of approximately one and a half months. The methodology described by González-Gaona and Sánchez-Martínez (2018) was used to collect larvae, and site collection coordinates, host species, height data, diameter, and degree of defoliation of the host were recorded. Branches with larvae were cut and stored in bags, coolers or individual flasks, transported to the laboratory and kept at room temperature. To determine the first instar, a sample of needles with eggs was monitored until the larvae hatched. Sixty larvae were also extracted from cocoons collected in the field. All larvae were sacrificed in warm water and preserved in 70 % ethyl alcohol. Each vial was labeled with the date, host, site and collector's name.
Since daily monitoring of larvae collected in the field to record molt changes and obtain each instar was not, the width of the cephalic capsule of 452 larvae of different instars was measured. Data were fitted to a normal distribution using PROC UNIVARIATE of SAS v. 9 statistical software (Villa-Castorena & Catalán-Valencia, 2004).
To determine the limits of two successive instars, the probability that larvae entering rank i actually belong to i + 1 was equated with the probability that larvae in rank i + 1 actually belong to rank (Got, 1988).
: = mean value of the larval head width for a specific instar
𝞼 = standard deviation of larval head width for a specific instar.
Cocoon measurements
A total of 40 whole sawfly cocoons were photographed, measured and described using Leica Application Suite V 4.6.1® software and a Leica DFC 295® camera mounted on a Leica M80® stereo microscope. Data was analyzed using descriptive statistics (mean and standard deviation) with SAS v. 9 statistical software.
Adult collection, description and measurement
Most of the adults were collected during field trips and the remaining ones were specimens emerged from cocoons in the laboratory. Some of the adults were dry mounted and some were preserved in 70 % ethyl alcohol. Observations and morphological description were made at the Laboratorio de Sanidad Forestal y Agrícola del Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Pabellón Experimental Field (CEPAB), Aguascalientes, Mexico.
Since key morphological characteristics in the identification of Diprionidae species are present in adult females (Smith et al., 2012), 34 dry-mounted females were used to determine the species. Additionally, one male specimen was included for general description purposes, knowing that Diprionidae males do not have morphological characteristics that allow for easy species differentiation.
Specimens were described morphologically using the taxonomic keys of Smith (1971, 1974, 1988) and Smith et al. (2012). The ovipositor (lancet) was removed from 29 females. First, the last abdominal segments were separated from the body and placed in 10 % potassium hydroxide (KOH) at 80 °C for 15 min to macerate the soft tissues and then rinsed with distilled water to remove residual KOH. Subsequently, the segments were dehydrated in 70 % ethyl alcohol for one minute and then another minute in 96 % ethyl alcohol; finally, they were preserved in 70 % ethyl alcohol. The abdominal segments were placed in a Petri dish with a drop of water and under a Motic SMZ-140® stereo microscope (magnification 10x, 20x and 40x) the ovipositor was extracted and mounted in Canada Balsam on a slide.
The characteristics observed in the ovipositors were number of rings and orientation, relative size of teeth in rings, and shape of serrulae. Appearance of ovipositors was compared with illustrations in Smith et al. (2012) and Smith (2019), and with lancets of Zadiprion specimens from the forest insect collection of the Pabellón Experimental Field, INIFAP.
Biological cycle
For information on developmental times, young larval instars were collected during the study period and kept in glass boxes (40 x 40 x 40 x 40 cm), labeled with codes (“Y” and “Z”). Larvae were fed with foliage from the same tree where they were collected. The boxes were sealed with tulle cloth and a spring band to favor air circulation and were kept in a cool, shaded place until cocoon formation and adult emergence.
Evaluation of field-collected cocoons
From August 24-26, 2020, we placed five sampling units of 1 m2 in each of 11 circular sites of 1 000 m2 (N = 55 sampling units). These units were established near defoliated trees at the sites to capture the greatest number of cocoons. The site was delimited, the leaf litter was removed until the soil was visible and dug to find the cocoons. These were collected and stored in bags, one per sampling unit, with their collection data. At the laboratory, the cocoons were individually checked to evaluate their condition and classified into one of the following groups: 1) with a wide, uniform cut at one end denoting adult sawfly emergence, 2) with lateral perforations corresponding to parasitoid emergence, and 3) torn by predators. It was accepted that these cocoons were formed the previous year and corresponded to the current generation of adults that oviposited in 2020.
Results
According to the taxonomic keys used as references, it was determined that the specimens studied are of a new species, which is described below.
Zadiprion llanderalae nov. sp. Sánchez-Martínez & Cibrián-Tovar
Etymology
Named after Celina Llanderal Cázares, entomologist and research professor at the Colegio de Postgraduados.
Adult female
Female is 9 to 13 mm long (mean = 10.2 mm, n = 34). Female head is light brown with a black spot above each antenna; light or only slightly dark postocellar area; lateral postocellar grooves are yellow on the dorsal side and dark on the occiput. Antennae are serrate with 22 light brown flagellomeres; the last six to eight are dark ventrally. The pronotum is light brown, including the propleuron. The mesoscutum is light brown; the dividing line of the lobes (left and right) is black and thin on the anterior and middle part, and dark on the posterior part. The mesoscutum is light brown, except in the inner basal part where it is black; the mesoscutum grooves (notauli) are light without black lines. The mesoscutum is light brown, dark on the posterior margin. The groove separating the mesoscutum from the mesoscutellum is black and markedly deep. Metanotum dark brown with black anterior and posterior margins (Figure 2A). Mesepisternum light brown with an oval, dark or black spot medially, occasionally barely visible and sometimes extending to the dorsal margin. Mesepisternum and mesosternum are light brown (Figure 2B). Forewings are yellow, transparent (amber-like), with a slightly dark hue in the radial cell and stigma; veins C, R, RS and M are transparent yellow (Figure 2C). The hind wings are yellow, transparent and with venation of the same color (Figure 2D). Abdomen predominantly black in dorsal view; tergites I light brown, tergites II to VIII black, with whitish stripes posteriorly when expanded and whitish in lateral view. Sternites dark anteriorly and whitish posteriorly. Legs have the puviliar pads on the basal tarsus of the hind leg nearly as long as or longer than half of the basal tarsus (Figure 2E). The lancet has nine rings: first ring absent, rings 2 and 3 slightly divergent and 4 to 10 subparallel; second ring with 11 to 13 distinguishable teeth; the central three larger than the rest and the last ring short. Basal serrula flat at apex, twice as wide as second serrula and slightly acute posteriorly. Serrulae 2 to 5 are flat at the apex and noticeably acute posteriorly (Figure 2F).
Figure 2 Female of Zadiprion llanderalae nov. sp. A) dorsal view (head and thorax), B) lateral view with the characteristic spot of the species on the mesepisternum (arrow), C) anterior wing, D) posterior wing, E) metathoracic legs and F) lancet with nine rings. Reference samples deposited in the forest insect collection of INIFAP, Pabellón Experimental Field.
Adult male
The male measured 8.8 mm in length (n = 1) and is black in appearance with thin, barely noticeable stripes on the posterior part of the tergites in dorsal view. The antennae are bipectinate composed of 27 flagellomeres, plus the scape and pedicel; the last seven flagellomeres unipectinate. The thorax is black with dark transparent wings, black legs with yellow segments on the tibia and trochanter and black femur (Figures 3A-3C).
Figure 3 Male of Zadiprion llanderalae nov. sp. A) dorsal view, B) frontal view of head and C) lateral view.
Zadiprion llanderalae nov. sp. differs from Z. falsus by the less robust appearance of the female and the smaller but more defined black spot on the mesepisternum. The lancet of Z. llanderalae has nine rings with the first ring absent, while Z. falsus has 11 rings with a larger number of teeth on the second ring and Z. howdeni has a short lancet with seven rings. Table 1 compares the lancets of the Zadiprion species with which Z. llanderalae was mistaken.
Table 1 Comparison of lancets of Zadiprion llanderalae, Z. falsus and Z. howdeni.
| Female | Lancet | Reference |
Zadiprion llanderalae
|
nine rings 
|
This document |
Zadiprion falsus
|
11 rings 
|
Smith (1988) Reproduced from Smith et al. (2012) under the fair use principle. |
Zadiprion howdeni
|
seven rings 
|
Reproduced from Smith et al. (2012) under the fair use principle. |
Material type
Holotype: female labeled “México/Oaxaca/Santa Catarina Ixtepeji/Paraje El Paredón and Lilexy, at an elevation of 2 483 m, 17° 11’ 26” N, 96° 35’ 5” W. 02/VII/2021, col. Guadalupe Gómez M., det. G. Sánchez Martínez 2021, field collected on Pinus oaxacana”, deposited in the INIFAP forest insect collection, Pabellón Experimental Field (CEPAB), Pabellón de Arteaga, Aguascalientes.
Paratypes: 15 ♀ with the same data as contained on the holotype label; 9 ♀ labeled “México/Oaxaca/Santa Catarina Ixtepeji/Paraje El Paredón, at an elevation of 2 385 m, 17° 11’ 19” N, 96° 35’ 07” W. 28/VII/2021, col. Sánchez Martínez, G., det. G. Sánchez Martínez 2021, field collected on Pinus oaxacana”, deposited in the insect collection of CEPAB; 10 ♀, 1 ♂ labeled: “México/Oaxaca/Santa Catarina Ixtepeji/Paraje La Cooperativa 17° 08’ 02.7” N, 96° 33’ 20.6” O. 2 632 m de elevación. 29/VII/2021, col. A. Hernández C., det. G. Sánchez Martínez 2021, Pinus oaxacana”, deposited in the insect collection of the Division of Forestry of the Universidad Autónoma Chapingo.
Eggs
Eggs inserted in the needles of the last year of growth, contiguous in position, in rows of 20 to 35; the female uses several contiguous needles to deposit them (Figure 4). Newly laid eggs are translucent white, turning light beige as they mature. They measure 1.07 mm in average length (maximum 1.2 mm, minimum 0.84 mm, n = 78) and 0.37 mm in average width (maximum 0.54 mm, minimum 0.29 mm, n = 78).
Larva
The frequency distribution of the cephalic capsule width data of 475 larvae suggested the existence of six larval instars (Table 2), which agrees with that indicated by Smith (1993) for sawflies; however, the division between instars was not clear, mainly because of the wide variation in the width of the cephalic capsule of larvae that recently formed cocoons.
The estimated parameters of the normalized data for each instar are shown in Table 2; the P values obtained in the Shapiro-Wilk test were close to 1, indicating a good fit of the data with respect to the normal distribution function. The limits of the ranges for each instar were obtained with the methodology of Got (1988) (Table 3).
Table 2 Estimated parameters of normal distribution functions of cephalic capsule width of Zadiprion llanderalae nov. sp. larvae by instar.
| Instar | N | Average (mm) | Minimum value | Maximum value | Shapiro-Wilk (P) |
|---|---|---|---|---|---|
| 1 | 69 | 0.438 ± 0.024 | 0.360 | 0.488 | 0.977 |
| 2 | 129 | 0.600 ± 0.061 | 0.506 | 0.750 | 0.874 |
| 3 | 34 | 0.867 ± 0.057 | 0.765 | 0.950 | 0.947 |
| 4 | 80 | 1.075 ± 0.070 | 0.920 | 1.246 | 0.990 |
| 5 | 74 | 1.390 ± 0.080 | 1.263 | 1.545 | 0.957 |
| 6 | 89 | 1.659 ± 0.081 | 1.552 | 1.849 | 0.929 |
± standard deviation of the mean.
Table 3 Range limits of cephalic capsule width of Zadiprion llanderalae nov. sp. larvae by instar.
| Instar | Head width (mm) |
|---|---|
| 1 | < 0.483 |
| 2 | > 0.483 to 0.738 |
| 3 | > 0.738 to 0.960 |
| 4 | > 0.960 to 1.223 |
| 5 | >1.223 to 1.523 |
| 6 | >1.523 |
Newly emerged larvae from eggs are almost transparent and pale (Figure 5A). As they feed, they become greenish, resulting from the ingestion of green foliage accumulating in the gut. Larval behavior is highly gregarious. Second to fourth instar larvae are green, form compact groups, have a light brown head and a round black eyelid (Figures 5B and 5C). Mature fifth and sixth instar larvae lose some of their gregarious behavior (Figure 5D), measure on average 2.6 cm in length (maximum 3.7 cm, minimum 1.4 cm, n = 163), the head is light brown with a round, black eyecup, and the thoracic and abdominal segments are almost smooth with short spines on the annular segments. The body is semitransparent and opaque, revealing green intestinal contents from consuming needles, with a broad dorsal light stripe (separated by a thin grayish stripe) and medium wide, grayish lateral stripes, often discontinuous along the body. The legs are whitish on the soft parts, with yellowish-brown sclerites on the coxa, trochanter, femur, tibia and tarsal nails.
Sawfly cocoons
Of the 40 sawfly cocoons measured, two groups were identified, small and large, the first measuring 3.6 mm in average length and the second 4.2 mm in average. The newly formed cocoons are whitish and then acquire a golden-brown color. In the inside, the larvae enter a resting stage until the pupa is formed.
Biological cycle
In the second half of April 2021, a few females resting on needles were collected, but no egg-laying were found; in May, no adults or egg-laying were recorded. The first ovipositions were recorded in the first week of June 2021, being more frequent in the following weeks and until the end of July; females were always observed guarding the egg rows. The first larvae were observed from the last week of June and were more abundant in the first week of July. Intermediate instars were present from July to October. Mature larvae were found from August, being more abundant from September to November; in this last month, they descend from the tree canopy to bury themselves in the ground and form their cocoon, where they will pass to the pupal stage. In December 2021 and January and February 2022, mature larvae on foliage were scarce. On the ground, the first sawfly cocoons were found since August; during September and until the first half of July of the following year they were more abundant, and in the second half of July there were fewer individuals. These observations indicate that an annual generation occurs with developmental stages present in several months. Figure 6 shows the times at which each developmental stage occurs. Field inspections determined that the larvae fed on needles of P. oaxacana, P. patula and P. leiophylla, with the former as the main host.
Figure 6 Development stages of Zadiprion llanderalae nov. sp. in Santa Catarina Ixtepeji, Sierra Norte de Oaxaca.
An average density of 39.2 ± 9.69 sawfly cocoons∙m-2 was recorded in 2019. Of the 2 156 cocoons collected in the 55 1 m2 sites, 1 081 cocoons (50.13 %) had parasitoid emergence holes and 810 cocoons (37.57 %) had a clean cut at one end, where females and males of Z. llanderalae were expected to emerge, of which 50 % (405) were females, i.e., 7.3 ± 1.80 females∙m-2. The remaining 265 sawfly cocoons (12.29 %) were closed; from some, in the months of November and December, adults of parasitoid wasps of the genus Exenterus (Hymenoptera: Ichneumonidae) emerged, which has been recorded in cocoons of Z. falsus (Cibrián-Tovar et al., 1995; González-Gaona & Sánchez-Martínez, 2018). In the cocoons remaining closed, we found remains of larvae or pupae with mycelium with white or greenish-gray coloration, probably from entomopathogenic fungi.
Discussion
For the Sierra Juárez, CONAFOR identified the species Z. falsus and Z. howdeni as the agents causing defoliation (Aguirre-Hidalgo et al., 2020; González-Gaona & Sánchez-Martínez, 2018; Suárez-Mota et al., 2018). In addition to the above, Aguirre-Hidalgo et al. (2020) mention Z. howdeni as the species present in Ixtlán de Juárez and indicate that the specimens they studied had a short ovipositor with nine rings and the first one absent. The morphological characteristics of the females and ovipositor of Z. howdeni, indicated by Smith (1988), are inconsistent with the characteristics of the specimens collected at the study site, which led to the identification of a new species: Zadiprion llanderalae nov. sp. Furthermore, the characteristics of the specimens studied by Aguirre-Hidalgo et al. (2020) conform to those described for Z. llanderalae nov. sp., suggesting that it is this species rather than Z. howdeni.
The life history data reported by Aguirre-Hidalgo et al. (2020) do not agree with that observed in this study. They report adult insects from February, eggs from April-August, larvae from the end of May-November and cocoons until the end of February. Meanwhile, the results of this research show the presence of adults from April, eggs from June to July, this being the shortest development stage, and larvae from the end of June to the beginning of December. Because of the different type of sampling used in the studies, further research is needed to gather more detailed data.
The importance of natural biological control by parasitoid wasps is evident, since 50 % of the cocoons were found under this condition, with wasps of the genus Exenterus (Hymenoptera: Ichneumonidae) being one of the parasitoids detected. In Mexico, sawfly parasitoids are reported in species of the families Tachinidae, Ichneumonidae, Braconidae, Perilampidae, Pteromalidae and Eulophidae (Cibrián-Tovar et al., 1995; González-Gaona & Sánchez-Martínez, 2018; Ruíz-Cancino & Khalaim, 2015). For Z. falsus, 30 % parasitism of Lamachus, Stylocriptus, Endasys y Exenterus (Hymenoptera: Ichneumonidae) and Spathimeigenia mexicana (Diptera: Tachinidae) have been found (Cibrián-Tovar et al., 1995; González-Gaona & Sánchez-Martínez, 2018).
Suárez-Mota et al. (2018) report an average density of 35 to 138 cocoons∙m-2, while that reported by Aguirre-Hidalgo et al. (2020) in the region of Ixtlán, Oaxaca, was 37.92 ± 8.63, which is similar to the 39.2 ± 9.69 of the present study in 2020 in Ixtepeji, Oaxaca.
Conclusions
The defoliator causing high infestations in the pine forests of Santa Catarina Ixtepeji in the last five years is a new species that in this study has been named Zadiprion llanderalae nov. sp. The biological cycle of Z. llanderalae is annual. The period of presence of eggs is short, while the larval and cocoon periods are the longest, which is important to know to plan measures to combat this pest.
