Effects of adults body size and larvae diet on the fecundity and percent fertility of eggs laid by Xylotrechus arvicola (Coleoptera: Cerambycidae) females, insect pest in Spanish vineyards
Abstract
Xylotrechus arvicola is a pest of grape in some vine-producing regions of the Iberian Peninsula. Biological parameters and relationships (fecundity and percent fertility of eggs in relationship to body size) of females obtained in the laboratory and captured in vineyards were studied. In laboratory conditions, the mean developmental time of larvae ranged from 384 to 392 days and pupal stage varied between 12 to 14 days. Body size (BS) of X. arvicola females was significantly bigger than males. Fecundity was greater in the laboratory (147 eggs) than in the field (50 eggs) females, but the percent fertility of the laboratory eggs was lower (16 eggs). Laboratory females showed a bigger relationship between the production of eggs and BS than females captured in vineyards. Wild females (PDO Ribera del Duero and Tierra de León) had a positive relationship between the percent fertility of eggs and the BS. No correlation between the percent fertility of eggs and the BS was displayed by females captured in PDO Toro, but these females had a higher percent fertility (53 eggs) than the others PDO's. These biological parameters and relationships studied suggest that the artificial diet may lack certain essential nutrients that vine varieties can provide that favor the fertility of eggs. This explains why wild females have the potential to become a problem pest in the Tempranillo grape variety, with bilateral cordon and bush vines training systems that have the highest incidence of this cerambycid.
Introduction
Xylotrechus arvicola is a xylophagous polyphagous that has become an important pest in vineyards (Vitis vinifera) in the main Iberian wine-producing regions, such as: La Rioja Alta and Alavesa (Ocete & Del Tío 1996; Ocete & López 1999), Navarra (Ocete et al. 2002), Castilla–La Mancha (Rodríguez & Ocaña 1997) and Castilla y León (Ocete & López 1999; Peláez et al. 2001), but it has also been identified in Prunus spinosa L. plantations (Biurrun et al. 2007). X. arvicola adults can measure between 8 and 20 mm in length (Moreno et al. 2003), with females exhibiting a total larger length than males (Rodríguez-González et al. 2016b).
After mating, X. arvicola females lay eggs that gather in the cracks or under the rhytidome of vine wood. The fecundity and the percent fertility of eggs extend over a long period of time (Rodríguez-González et al. 2016a). The location of the eggs enables the emerging larvae to gain entrance into the wood and make galleries inside the plant. Therefore, the most susceptible stages of the species to insecticide treatments are adults, eggs and neonate larvae. Moreover, once inside the wood, the larvae are protected from chemical compounds and the treatment on adults is also problematic due to a pattern of emergence that is staggered in time (García-Ruiz 2009). Soria et al. (2013) reported the emergence period between late June and mid-July in vineyards of La Rioja, Moreno (2005) from March to end-July in vineyards of Valladolid, whereas Biurrun et al. (2007) determined the period between 14 May and 26 August for plantations of Prunus spinosa L in Navarre.
The life cycle of X. arvicola has been monitored by various authors (Vives-Noguera 2000; Moreno 2005; García-Ruiz 2009; García-Ruiz et al. 2011, 2012; Rodríguez-González 2014) and it has been suggested that, in field conditions, larval development lasts 2 years for different Spanish wine-producing regions. However, such estimation has not been experimentally tested. In a previous work, Moreno (2005) reared X. arvicola from neonate larvae to adult, and pointed out that neonate larval mortality was the most important constraint to rearing this species in the laboratory. To make rearing X. arvicola easier under laboratory conditions, García-Ruiz et al. (2012) evaluated four different diets, out of which, the Semi Synthetic Iglesias (SSI) diet proposed by Iglesias et al. (1989) for the artificial rearing of coleoptera lignicolas under laboratory conditions, had a survival rate of 36%.
Once an acceptable method of rearing X. arvicola through SSI diet under laboratory conditions was known, Rodríguez-González (2014) obtained a population of X. arvicola adults, from which various tests were conducted: (i) By studying the reproductive patterns, females obtained in the laboratory were shown to have higher fertility than females captured in the vineyard (Rodríguez-González et al. 2016a); (ii) Another test performed on these adults was a biometric study, which showed that adults reared in the laboratory have a greater total body size than adults captured in the field (Rodríguez-González et al. 2016b). Moreover, a positive relationship between pupal weight and fecundity has been demonstrated in other cerambycid species (Jikumaru et al. 1994; Smith et al. 2002; Wang et al. 2002). In addition, greater weight also means greater body size in cerambycids.
Consequently, our aims for conducting this study were: (i) to investigate the relationship between body size and fecundity and percent fertility in X. arvicola females; and (ii) to determine the effects of diet on the fecundity and percent fertility of eggs laid by X. arvicola females that originated from larvae subjected to different diets (vine wood of the natural host and artificial diet in the laboratory).
Materials and methods
Insects groups and origin
- Group 1 (Field). Xylotrechus arvicola wild adults were captured during 2010, 2011 and 2012 through an interception trap (CROSSTRAP) in three vineyards located in wine-producing regions of the Iberian Peninsula with Protected Designation of Origin (PDO) (PDO Ribera Del Duero, PDO Toro and PDO Tierra de León) and obtained from infected wood during 2010 and 2011 in the PDO Ribera Del Duero. To visit the traps and send the X. arvicola adults captured to the laboratory, the methodology described by Rodríguez-González et al. (2016b) was used. To obtain X. arvicola adults from infested vine wood, the wood was cut and taken to the laboratory and was introduced into plastic boxes (55 × 40 × 35) (length × width × height) and covered by mesh. The adults that emerged from the wood were captured and matched with other X. arvicola adults from the same origin (infected wood). More details about the vineyards where the X. arvicola adults were captured are shown in Table 1.
- Group 2 (Laboratory). Xylotrechus arvicola adults obtained in laboratory from a population of X. arvicola larvae reared through the SSI diet (Iglesias et al. 1989) from eggs of wild X. arvicola females captured in the vineyards within the described PDOs. To rear each larva using the SSI diet, the methodology described by García-Ruiz et al. (2012) was used. Sex identification was performed after the complete esclerotization and melanization of the adults. Once the fatty abdominal reserves were reabsorbed, it was possible to distinguish different body coloration between males and females as described by Moreno (2005). Then, females obtained through the SSI diet were paired with males obtained through the SSI diet. If a male died, another one was added to allow females to continue laying eggs (García-Ruiz et al. 2012).
| Details | PDO Ribera del Duero | PDO Toro | PDO Tierra de León |
|---|---|---|---|
| Location, Province | Peñafiel, Valladolid | El Pego, Zamora | Gordoncillo, León |
| Coordinates | 41°35′39.1″N - 4°05′19.1″W | 41°20′26.4″N - 5°25′51.8″W | 42°08′14.9″N - 5°25′41.6″W |
| Training system of vines | Bilateral cordon | Bush vines | Bilateral cordon |
| Training system characteristics | Spur pruning over two arms per trunk (1 m) | Spur pruning over 4–5 branches per trunk (0.5 m) | Spur pruning over two arms per trunk (1 m) |
| Vitis vinifera variety | Tempranillo | Tempranillo | Tempranillo |
| Vines were spaced (m x m) | 3 × 1.5 | 3.5 × 3.5 | 3 × 1.5 |
| Age (years old) | 25 | 50 | 18 |
| Years of capture in vineyards | 2010, 2011 and 2012 | 2011 and 2012 | 2012 |
| Number of adults evaluated | 55 males and 32 females | 53 males and 26 males | 51 males and 36 females |
Environmental conditions
- Larval development time. Developmental time of each stage (the amount of time that an individual was a larva, a prepupa and a pupa) in larvae that successfully completed their larval cycle and reached the adult stage (adults obtained in laboratory) were recorded for Group 2.
- Adult Longevity. In Group 2, the longevity was also recorded for both sexes of adult insects obtained in laboratory (daily since the end of their pupal stage until the last adult died).
- Fecundity. To determine the oviposition of X. arvicola females in Group 1, 32 females captured in the PDO Ribera del Duero were paired individually with 55 males (10 of these adults, obtained from the infected wood, 5 females and 5 males, were paired between them, one female with one male), 26 females captured in the PDO Toro were paired individually with 53 males, and 36 females captured in the PDO Tierra de León were paired individually with 51 males. Similarly, 31 females obtained in the laboratory were paired with 51 males to assess the fecundity within Group 2. The oviposition substrates were reviewed daily and the numbers of eggs laid were recorded. The eggs were extracted and placed in 55 mm diameter Petri dishes. The plates were covered with aluminium foil to ensure hatching in complete darkness. Eggs hatched 7–8 days after oviposition and collection dates were noted. The neonate larvae were extracted daily with the help of a brush and transferred to plastic cylindrical containers with the diet. If a X. arvicola male died, another male was added to allow females to continue laying eggs (the females always had to have a male because the eggs laid by the female had to be fertilized, females without a male can lay eggs, but the eggs are not fertilized). Only one female and one male were introduced at a time, since in the laboratory it has been observed that several insects of the same sex in the same glass jar end up killing one another. The glass jars were checked daily until the last female died (both wild and laboratory).
Biometric trait studied (Body Size)
Once adult insects died, their bodies were measured. The biometric trait studied was the body size (BS; obtained from the sum of elytra length and pronotum length) (Fig. 1) to have a better knowledge of the X. arvicola body size. This biometric trait was evaluated on 334 adults with the help of a magnifying glass and Motic Software 2.0.
Statistical analysis
Analysis of covariance (ANCOVA) was used to examine the effect of BS of the X. arvicola female (fixed factor) on fecundity and percent fertility of eggs as a covariate (Sokal & Rohlf 1995). The data of the remaining biological aspects was evaluated using analysis of variance (ANOVA) followed by a Tukey's honest significant difference (HSD) test. The alpha level was P ≤ 0.05. Analyses were conducted using SAS Software 9.1.2 (SAS Institute Inc., 2004, Cary, NC, USA).
Results
Larval development time
No significant differences were found between sexes for any of the immature developmental times of X. arvicola pertaining to Group 2 (Table 2).
| Group | n | Sex | Larval development time (days) | Prepupal stage (days) | Pupal stage (days) |
|---|---|---|---|---|---|
| 2 | 51 | Male | 384.15 ± 3.43a | 7.02 ± 0.29a | 12.78 ± 0.63a |
| 31 | Female | 392.19 ± 3.75a | 7.29 ± 0.61a | 14.32 ± 0.72a |
- Means followed by the same lowercase letter in columns were not significantly different between sexes for the same group (ANOVA followed by Tukey's HSD, P > 0.05).
BS and adult longevity
Males obtained in the laboratory showed a greater BS, which was significantly different from males captured in the different vineyards. Males from the PDO Toro exhibited the greatest BS, although it was not significantly different from the other males captured (Table 3). Once again, females obtained in the laboratory had a greater BS, which was significantly different from females captured from the three vineyards. Of those, the females originating from the PDO Toro showed the greatest BS, in spite of not being significantly different from that of the other females captured (Table 3). Nonetheless, significant differences were found between sexes of adults obtained in the laboratory, and captured in vineyards from PDO Ribera del Duero, PDO Toro and PDO Tierra de León. For both group and origin, females showed greater BS than males (Table 3). For adults obtained in the laboratory, no significant differences were found between sexes in relation to the longevity (Table 3).
| Group | Origin | N | Sex | BS (mm) | Longevity (days) |
|---|---|---|---|---|---|
| 1 | PDO Ribera del Duero | 55 | Male | 10.03 ± 0.17bB | 15.01 ± 1.83* |
| 32 | Female | 10.71 ± 0.28bA | 24.06 ± 4.10* | ||
| 1 | PDO Toro | 53 | Male | 10.58 ± 0.20bB | 14.39 ± 1.56* |
| 26 | Female | 11.67 ± 0.38bA | 17.80 ± 3.61* | ||
| 1 | PDO Tierra de León | 51 | Male | 10.04 ± 0.15bB | 20.23 ± 1.59* |
| 36 | Female | 10.91 ± 0.29bA | 21.25 ± 1.98* | ||
| 2 | Laboratory | 51 | Male | 14.94 ± 0.26aB | 28.56 ± 2.70A |
| 31 | Female | 15.78 ± 0.14aA | 36.83 ± 3.94A |
- Means followed by the same lowercase letter in the column were not significantly different among different groups and origin for the same sex. Means followed by the same capital letter in the column were not significantly different between sexes for the same group and origin (ANOVA followed by Tukey's HSD, P > 0.05).
- * Age of X. arvicola adults since his capture in vineyards. The adult age previous to be captured in vineyards was unknown.
Fecundity
The total number of eggs laid per X. arvicola female obtained in the laboratory was significantly higher than that of females captured in vineyards (through an interception trap in the vineyards or obtained from infected wood). Even though PDO Tierra de León showed the highest number of total eggs laid per female, no significant differences were found among females captured from the remaining vineyards (Table 4). The percent fertility of eggs per X. arvicola female captured from PDO Toro was higher than that of other females, not being significantly different from the percent fertility of the eggs laid by the remaining wild females evaluated but significantly different from females obtained in the laboratory (Table 4). The number of unhatched eggs per X. arvicola female obtained in the laboratory was significantly higher than females captured in vineyards. Specifically, the females originating from PDO Tierra de León exhibited the highest number of unhatched eggs, although the comparison among females captured in the vineyards was not significantly different (Table 4).
| Group | Origin | n | Total eggs | Percent fertility of eggs | No. of unhatched eggs |
|---|---|---|---|---|---|
| 1 | PDO Ribera del Duero | 32 | 50.87 ± 4.33b | 43.11 ± 5.31a | 27.06 ± 3.39b |
| 1 | PDO Toro | 26 | 51.76 ± 6.97b | 53.40 ± 7.63a | 22.69 ± 5.14b |
| 1 | PDO Tierra de León | 36 | 69.33 ± 6.27b | 45.98 ± 5.29a | 42.11 ± 6.20b |
| 2 | Laboratory | 31 | 147.71 ± 15.92a | 16.69 ± 3.16b | 126.80 ± 15.65a |
- Means followed by the same lowercase letter in each column were not significantly different (ANOVA followed by Tukey's HSD, P > 0.05).
Relationship between BS and eggs laid per female
Fecundity (total number of eggs) as function of the BS between females obtained and captured was not significantly different. Regardless of the origin of the adult insect, the fecundity was positively correlated with their BS (Fig. 2). Females obtained in laboratory showed the highest relationship between their fecundity and BS although the correlation was not significantly different from females captured in the three vineyards. Among the females captured, there were no significant differences in fecundity as a function of the BS, however, the females from PDO Tierra de León had the highest fecundity–BS relationship, followed by females from PDO Ribera del Duero, and PDO Toro.
Percent fertility (percentage of eggs hatched) as function of the BS between females obtained in the laboratory and captured in vineyards was significantly different (F = 16.84; df = 3,120; P = 0.0001). A positive relationship between the percent fertility of egg and the BS was found for PDO Ribera del Duero and PDO Tierra de León environments. The correlation displayed by the females obtained in laboratory was reversed in females captured from the vineyards. No correlation between the percent fertility of eggs and the BS was displayed by females captured in PDO Toro, but these females had a higher percent fertility (53 eggs) than the other PDOs. (Fig. 3).
Discussion
Females that emerged from larvae reared in the laboratory of other cerambycid species are reported to be significantly heavier than males, such as Acalolepta vastator Newman (Goodwin & Pettit 1994), Anoplophora glabripennis Motschulsky (Dubois et al. 2002) or Oemona hirta F. (Wang et al. 2002). A positive correlation between fecundity and female size is widespread in insects (Honěk 1993) irrespective of nutritional background (Torres-Vila et al. 1999) and specifically in cerambycid beetles (Iwabuchi 1988; Wang et al. 2002; Kato et al. 2000; Torres-Vila et al. 2016). Moreover, a positive relationship between pupal weight and fecundity has been demonstrated in other cerambycid species (Jikumaru et al. 1994; Smith et al. 2002; Wang et al. 2002). In addition, greater weight also means bigger size and/or bigger body size in cerambycids. The present investigation reveals that the fecundity of females born from individuals reared with the SSI diet is greater than that of females captured in different vineyards. This suggests that the SSI diet satisfies larval nutritional requirements. Fecundity values exhibited by X. arvicola females were higher than other cerambycids, such as Xylotrechus quadripes (103 total eggs per female) (Visitpanich 1994), Aeolesthes sarta (123 total eggs per female) (Mazaheri et al. 2007) and Enaphalodes rufulus (80 total eggs per female) (Galford 1985). Nonetheless, the fecundity results of this paper were lower than those obtained by García-Ruiz et al. (2012) for X. arvicola, who reported a fecundity of 244 and 196 eggs per female obtained in laboratory and emerged from infected wood (Vitis vinifera) respectively, but greater than those obtained by Rodríguez-González et al. (2016a) with a similar type of X. arvicola females in other experiments.
The females needed more time to complete their larval development time, which may explain why males usually emerge earlier than females, in environments, field (García-Ruiz 2009) and laboratory (Wang et al. 2002). This phenomenon, known as protandry, is a strategic evolution of insects so that when females appear, there are enough males to ensure the copulation and perpetuate the species. For instance, under laboratory conditions, the larval development time of females is longer than males in Ataxia hubbardi whereas the opposite occurs in Mecas inornata as described by Rogers and Serda (1979).
The greater BS obtained in laboratory for both sexes of adult insects was visually observable during the rearing periods. Therefore, the laboratory conditions favored larger sizes in X. arvicola larvae, prepupae, pupae and adults (Rodríguez-González et al. 2016b). The final size of the insects reared in the laboratory reflected the volume of material that had been used as host (Michalcewicz & Ciach 2012). The controlled laboratory conditions of development in the laboratory in which larvae were kept in greater volume with artificial diet could result in laboratory adults being bigger than wild adults (Rodríguez-González et al. 2016b). However, these size variations in insect bodies may also occur depending on the host (plants, species and variety). For instance, the total length measures for X. arvicola adults in Prunus spinosa trees described by Biurrun et al. (2007) varied between 11.15 mm in males and 13.00 mm in females. X. arvicola adults captured in Vitis vinifera exhibited an average total length of 10.58 mm in males and 11.67 mm in females, which were smaller to those described by Moreno (2005) with 11.52 mm and 13.44 mm in males and females, respectively. Nonetheless, females obtained in the laboratory and captured in the field had a greater total length than males. A similar relationship between sexes was obtained by Moreno (2005) and (Rodríguez-González et al. 2016b), who also had similar values in the total length of X. arvicola males and females captured in vineyards and obtained in laboratory. The greater total length of females was also cited for other cerambycid beetles, such as Iberodorcadion fuliginator by Bahillo (1997) or Iberodorcadion (Hispanodorcadion) pseudomolitor and Iberodorcadion (Hispanodorcadion) mosqueruelense by González et al. (2001).
The longest life spans have been documented for species in the subfamily Lamiinae (Linsley 1959); with adults of Anoplophora glabripennis (112 days) (Keena 2005). Galford (1985) explained that individuals of the subfamily cerambycinae (to which X. arvicola belongs) have shown life spans that vary between 21 days for Enaphalodes rufulus (Coleoptera: Cerambycidae) adults and 29 days for Xylotrechus quadripes (Coleoptera: Cerambycidae) adults, as was described by Visitpanich (1994). The longevity results of the present investigation, for females obtained in laboratory (36 ± 3 days) were similar to those described by García-Ruiz et al. (2012), who reported a lifespan of 37 ± 4 days.
In conclusion, the results suggest that the SSI diet has more nutrients that favor the production of eggs per laboratory female (147 eggs), showing a bigger relationship between the production of eggs and the BS than females captured in vineyards. The percent fertility of eggs per wild female was higher than laboratory females and a positive relationship between the percent fertility of eggs and the BS was found in females captured in PDO Ribera del Duero and PDO Tierra de León. No correlation between the percent fertility of eggs and the BS was displayed by females captured in PDO Toro, but these females had a higher percent fertility (53 eggs) than the other PDOs. These biological parameters and relationships studied suggest that the artificial diet may lack certain essential nutrients that vine varieties can provide that favor the percent fertility of eggs. X. arvicola females need more time to complete development, which may explain why males usually emerge earlier than females in both environments (laboratory and vineyards). This explains that wild females have the potential to become a problem in the Tempranillo grape variety, with bilateral cordon and bush vines training systems having the highest incidence of this cerambycid.
Acknowledgments
The study was carried out thanks to a Project “Xylotrechus arvicola, técnicas de seguimiento y control en el cultivo de la vid” (VA/090137/S21), financed by the Rural Developmental Programme in Castilla y León-co-financed by the European Agricultural Fund for Rural Developmental (FEADER). The authors would also like to acknowledge the Department of Engineering and Agricultural Sciences of the Higher and Technical School of Agricultural Engineering and the Institute of Environment, Natural Resources and Biodiversity of the University of León.
