Factors limiting the northern distribution of the blueberry maggot , Rhagoletis mendax ( Diptera : Tephritidae ) in Eastern Canada

Until recently, the Canadian distribution of the blueberry maggot, Rhagoletis mendax Curran (Diptera: Tephritidae), was restricted to Nova Scotia, Prince Edward Island and New Brunswick. The insect was fi rst mentioned in southern Quebec in 1996 and, to date, it has not reached the Lac St-Jean region, where 34% of Canadian blueberry acreage is located. Two questions concerning the northern limit of distribution of the blueberry maggot in Quebec were addressed. First, are wild plants suitable hosts for larval development? We collected the fruit of fi ve wild plants, (e.g. Vaccinium corymbosum, Vaccinium angustifolium, Vaccinium myrtilloides, Gaylussacia baccata, and Aronia melanocarpa) growing in southern Quebec and allowed larvae to complete their development into pupae. Blueberry maggot pupae were recovered from Vaccinium corymbosum, Vaccinium angustifolium, and Gaylussacia baccata, indicating that these plants are suitable for larval development. Second, are harsh winter temperatures a factor limiting the northern distribution of the blueberry maggot? Pupae collected in Quebec and Nova Scotia were put in the soil in the fall and were brought back to the laboratory to determine their supercooling points at different times during winter. The supercooling points of pupae collected in Quebec and Nova Scotia averaged –22.6°C. In natural conditions, air temperatures <–20°C are frequently observed in Quebec in January, February and March. However, due to snow cover, soil temperatures are rarely <–12°C. If –22.6°C constitutes the lower limit for the survival, then winter temperatures are probably not a limiting factor to its northern distribution in Quebec, because blueberry maggot pupae overwinter in the soil.

Over the years, the blueberry maggot steadily extended its geographical distribution in Quebec and Ontario (CFIA, 2012).At the time of this writing, it has not yet reached the Lac St-Jean region, where most (ca.85% of managed surfaces) Quebec lowbush blueberries are produced without insecticide treatment (Ministère de l'Agriculture, des Pêcheries et de l 'Alimentation, 2011).As of 2010, Quebec were introduced individually in cavities of a microtube storage box (15 × 10 × 6.5 cm) in which a thermocouple (type T, size 36) allowed to maintain one pupa against a wall of a cavity (Fig. 1A).The thermocouple was connected to a data acquisition unit (model 34970A, Agilent Technologies®, Loveland, Colorado, USA) driven by the software Benchlink® (Agilent Technolo-gies®, Loveland, Colorado, USA) to take one temperature measurement per second.Cotton wool was placed in the lid of the storage box for thermal insulation.To further insulate the set-up, the storage box was placed in a styrofoam box (30 × 20 × 13 cm) (Fig. 1A), and the empty space between the two boxes was fi lled with vermiculite.The styrofoam box was put in a freezer set at -80°C until the SCP occurred (Fig. 1B, C).The set-up allowed to determine the SCPs of 15 blueberry maggot pupae at a time.Comparison of SCPs was done with an unpaired t-test with the software XLSTAT (Addinsoft, 2011).

RESULTS AND DISCUSSION
The number of pupae retrieved from fruit collected from fi ve wild plant species was highly variable.Assuming one larva per fruit, the percentage of fruit infested ranged from 0 to 18.4% in G. baccata (i.e., black huckleberry), 0 to 4.31% in V. angustifolium, 0 to 11.9% in V. corymbosum, and from 0 to 0.16% (2 pupae) in V. myrtilloides (i.e.velvet-leaved blueberry) (Table S1).Smith et al. (2001) found that G. baccata is a suitable host as indicated by high fruit infestation.They found highly variable % fruit point of R. mendax of pupae collected in Quebec and Nova Scotia in an attempt to determine if harsh winter conditions could be a limiting factor for blueberry maggot distribution in Quebec.

Suitability of wild hosts
Mature fruit of fi ve wild plant species growing in Quebec were collected in fi ve locations of southern Quebec (see dates and coordinates in Table S1 -supplemental material) reported to have blueberry maggot presence.For each location, fruit were counted, weighed and laid on fi ne sand to allow for the development of pupae.After 30 days, the pupae were extracted from the sand and counted.

Blueberry maggot material
Wild berries were collected in August 2004 and 2005 near the Ecological Reserve Pin Rigide, St-Chrysostome (45°05.603´N;73°52.117´W),Qc, Canada.In the laboratory, they were put at ca. 22°C (14L : 10D) and laid on moist sand (ca.85% sand and 15% water, by volume) where blueberry maggot pupated.Several weeks later, the sand was sifted to extract pupae.The extracted pupae were randomly assigned to groups, each approximately corresponding to a winter month (Table 2).In November 2004 and 2005, the groups of pupae were each placed in metallic sieves (8 cm diameter, 3 cm deep, 16 mesh size) that were buried ca. 3 cm in the soil of a commercial blueberry fi eld (located at St-Chrysostome: 45°06.878´N;73°49.980´W)documented to be infested by the blueberry maggot.Electronic temperature loggers (Optic Stowaway® Temp., Onset computer Corporation, Pocasset, MA, USA) were positioned at a depth of 3 cm in the soil nearby the sieves containing the pupae to monitor (one temperature measurement per hour) soil temperatures from November to May.As previously described, electronic temperature loggers were also positioned in non-infested commercial blueberry fi elds located in L'Épiphanie (45°49.130´N;73°31.562´W),Frelighsburg (45°08.190´N;72°51.707´W),Saint-Eugène (49°00.228´N;72°19.904´W),Normandin (48°46.754´N;72°33.272´W)and Labrecque (48°39.670´N;71°31.734´W),Qc, Canada.
Air temperatures at 1.5 m from ground level were obtained from weather stations belonging to the Quebec cooperative weather network (Lepage & Bourgeois, 2011).Stations located at Frelighsburg and Normandin provided data on site.Weather stations of Franklin (45°01.983´N;73°55.000´W)and L'Assomption (45°49.002´N;73°25.999´W)provided approximations of air temperatures for the nearby localities of St-Chrysostome and L'Épiphanie respectively.Air temperatures from 2003 to 2013 were extracted from a database with the software CIPRA (Bourgeois et al., 2008).
To determine if SCPs of blueberry maggot pupae in Quebec are similar to those of another region where blueberry maggot overwinters, fruit samples were collected in Nova Scotia from heavily infested commercial blueberry fi elds and processed as described previously.Pupae were stored at 2°C for several weeks, and shipped to the Horticultural Research and Development Centre at Saint-Jean-sur-Richelieu when needed for experiments.

Determination of SCP
Each month, a sieve containing one group of pupae was extracted from the fi eld (St-Chrysostome, Qc) and brought to the laboratory.Their SCP, i.e. exotherms caused by the release of heat of crystallization (Denlinger & Lee, 1998), was determined within ca.24 h.In order to have a slow decrease of temperature, the following set-up was developed.Blueberry maggot pupae  2), SCPs occurred at ca. 1100-1200 seconds and around -23.37°C; C -detailed view of temperatures determined between 1000 and 1400 s.
In northern Quebec the suitability of wild plants as hosts can be questioned because climatic conditions prevailing in that region are much harsher than those prevailing in Michigan, southern Ontario or southern Quebec (Table 1).For example, there are 130 frost-free days in Normandin, a locality of the Lac St-Jean region, while there are > 154 frost-free days in most of the localities sampled by Smith et al. (2001) (Table 1).In the fall at Normandin, the earliest and latest frosts occurred on 13 August and 25 September, respectively; on 10 September there is 50% probability that frost occurred (Ouellet & Laporte, 1963).In the fall at Saint-Clotilde (a site located ca. 10 km from St-Chrysostome), the earliest and latest frosts occurred on 25 August and 7 October, respectively; on 13 September there is 50% probability that frost occurred (Ouellet & Laporte, 1963).Furthermore, the annual average temperature in Normandin is 2.6°C, while it is > 6.1°C in most of the localities  sampled by Smith et al. (2001) in Michigan.Under colder conditions in the North, it is likely that the blueberry maggot larvae will have less time to complete their larval development, not only because lower temperatures directly slows developmental duration in poikilotherms, but also because lower temperatures indirectly decrease the duration of availability of suitable fruit to complete larval development.
The average SCPs of blueberry maggot pupae collected in Quebec (-22.6°C) and Nova Scotia (-22.6°C) were not signifi cantly different (t = -0.118,df = 1023, p = 0.906), while average standard errors were 4.12 (n = 11) and 4.29 (n = 10), respectively, and the maximal standard error was 7.08 (Table 2).In his review on insect cold hardiness, Bale (1996) stated that the SCP is simply the physical process of supercooling, but possibly the lower limit of survival in an overwintering insect.The determination of the lower limit requires formal experimentation.Partial determination has been done by Vincent et al. (2014) who found that exposure of blueberry maggot larvae and pupae at -20°C for > 2 days caused 100% mortality in laboratory conditions, while exposure at -20°C for 2 days did not cause 100% mortality.
Soil temperatures measured at a depth of 3 cm were frequently > -10°C during the winter months of 2005 and 2006 (Fig. 2).In our data set, soil temperature was lower than -20°C only once (-21.8°C) in 2003 at l'Epiphanie when snow cover was exceptionally rare.This is consistent with the data produced by Ouellet et al. (1975), who reported average monthly temperatures (taken at 1 cm in the soil) for 15 localities of the Lac St-Jean region.These average temperatures ranged from -1.1 to -6.9°C in January, the coldest month of the year.In contrast, from 2003 to 2013, minimum air temperatures measured at 1m above the soil in L'Assomption (locality near l'Epiphanie) were < -20°C 111 times in January, 70 times in February and 24 times in March (Table S2).Likewise, from 2003 to 2013, minimum air temperatures measured at 1m above the soil in Normandin were < -20°C 230 times in January, 173 times in February and 97 times in March (Table S2).Air temperature, used as input variables for the model of Dobesberger & Macdonald (1993), is a poor predictor of the temperature that the pupae might experience in the soil, where the temperature is moderated by the soil and snow cover in Quebec.

CONCLUSIONS
Two conclusions emerge from our study.First, several wild plant species are suitable hosts in southern Quebec (e.g.Ormstown, Qc), where the number of frost-free days and average temperature is comparable (Table 1) to those of several localities reported in Smith et al. (2001).It remains to be seen if these wild plants will be suitable hosts in the Lac St-Jean region.Other species can also be suitable host plants, including cultivated highbush and lowbush blueberry.In their study, Smith et al. (2001) found that G. dumosa is a valid host allowing pupation of the blueberry maggot, while no pupae were retrieved from Vaccinium pallidum Aiton.Second, the supercooling point of blueberry maggot pupae is -22.6°C.In natural conditions, soil temperature < -20°C is a rare mortality factor for blueberry maggot pupae because there is frequently snow cover that has insulating effect against air temperatures.Therefore, if -22.6°C constitutes the lower limit for the survival, then winter air temperatures probably do not represent a limiting factor to blueberry maggot northern distribution.2. The dotted vertical line represent the maximal value of the standard error associated with SCP of pupae, i.e. 7.08 (see Table 2).

Fig. 1 .
Fig. 1.A -set-up to determine the SCP of blueberry maggot pupae in the laboratory; B -determination of SCPs of blueberry maggot pupae occurred when a slight elevation of temperature (example shown by arrow) was measured.In the fi ve selected examples (extracted from the dataset 26 January 2006, Quebec; Table2), SCPs occurred at ca. 1100-1200 seconds and around -23.37°C; C -detailed view of temperatures determined between 1000 and 1400 s.

Fig. 2 .
Fig. 2. Soil temperatures measured at 3 cm depth in several localities of Quebec during winter 2005 and 2006.The dotted horizontal line represents the average SCP of pupae, i.e. -22.6°C.The two lines hovering around the dotted horizontal line are SCPs determined for blueberry maggot pupae of Quebec (empty bullets) and Nova Scotia (black triangles), as reported in Table2.The dotted vertical line represent the maximal value of the standard error associated with SCP of pupae, i.e. 7.08 (see Table2).

Table 1 .
Number of frost-free days and mean annual temperature in 6 locations infested with blueberry maggot and 2 non-infested localities of north eastern North America.

Table 2 .
Supercooling points (°C) of blueberry maggot pupae collected in Quebec and Nova Scotia in 2004 and 2005; n = number of pupae used.

Table S1 .
Suitability of wild fruit collected in Southern Quebec for larval development of the blueberry maggot.All locatities were at altitude ranging from 161 to 341 m from sea level.