Exploration for olive fruit fly parasitoids across Africa reveals regional distributions and dominance of closely associated parasitoids

The olive fruit fly, Bactrocera oleae, has been a key pest of olives in Europe and North America. We conducted the largest exploration for parasitoids associated with the fly across Sub-Saharan Africa (Kenya, Namibia, and South Africa) including some of the fly’s adjoining regions (Canary Islands, Morocco, Réunion Island and Tunisia). From Sub-Saharan regions, four braconids were collected: Bracon celer, Psytallia humilis, P. lounsburyi, and Utetes africanus. Results showed that their regional dominance was related to climate niches, with P. humilis dominant in hot semi-arid areas of Namibia, P. lounsburyi dominant in more tropical areas of Kenya, and U. africanus prevalent in Mediterranean climates of South Africa. Psytallia concolor was found in the Canary Islands, Morocco and Tunisian, and the Afrotropical braconid Diachasmimorpha sp. near fullawayi on Réunion Island. Furthermore, we monitored the seasonal dynamics of the fly and parasitoids in Cape Province of South Africa. Results showed that fruit maturity, seasonal variations in climates and interspecific interactions shape the local parasitoid diversity that contribute to the low fly populations. The results are discussed with regard to ecological adaptations of closely associated parasitoids, and how their adaptations impact biocontrol.


Results
Parasitoid regional distribution and diversity. Surveys from 110 sites of wild olives, O. europea nr.
ssp. cuspidata, in seven African regions yielded a total of 443,308 olive berries ( Fig. 1), from which 72,453 fly pupae, 27,848 adult B. oleae and 22,576 adult braconid parasitoids were obtained (Table 1). Two closely related African Bactrocera species, B. biguttula (Bezzi) (1.2%) and B. munroi White (2.6%) were also recovered, but in low numbers and with B. biguttula found only in South Africa and B. munroi only in Kenya (Table 1). Five Opiinae braconid wasps were recovered: Diachasmimorpha sp. near fullawayi, P. concolor, P. humilis (Szépligeti), P. lounsburyi and U. africanus; one Braconinae braconid wasp, B. celer, was also recovered. Psytallia concolor was the only species found in the Canary Islands, Tunisia and Morocco, whereas D. sp. near fullawayi was the only species recovered in the Réunion Island (Fig. 1). The other four species were found in Namibia and South Africa and three of them (except P. humilis) were found in Kenya, with P. lounsburyi, P. humilis and U. africanus being the predominant parasitoid species in Kenya, Namibia and South Africa, respectively (Fig. 1).
PCA revealed two major components that jointly explained 79.1% of the variance in the regional climatic variables (eigenvalues: component 1 = 3.76, 46.9% of variance; component 2 = 2.58, 32.2% of the variance) (Fig. 3). Despite the overlap of a few sites, the explored regions represented clearly different climate types. The climates in the Canary Islands, Morocco and Namibia were similar and are characterized by high temperatures and low precipitation. However, the climates in Kenya were related positively to the precipitation but negatively to the maximum temperature of the warmest month, with South Africa falling between these two climate types. The Scientific Reports | (2021) 11:6182 | https://doi.org/10.1038/s41598-021-85253-y www.nature.com/scientificreports/ climates of Réunion Island are characterized by high precipitation and marked thermal contrasts across the island. The regional dominance of the parasitoid species in the three Sub-Saharan countries was reflected in the PCA ordination (eigenvalues: component 1 = 5.62, 51.1% of variance; component 2 = 2.17, 19.7% of the variance) (Fig. 4). Sites in Kenya were assigned on the left while sites in Namibia were assigned on the right, and those in South Africa were in the middle. There was a positive relationship between the annual mean temperature or maximum temperature of the warmest month and the relative abundance of P. humilis, however this relationship was negative for P. lounsburyi. The relative abundance of U. africanus was negatively correlated with the minimum temperature of the coldest month and strongly associated with the precipitation during the wettest quarter.
Seasonal host-parasitoid dynamics. Independent of the regional surveys, more localized surveys were conducted at 24 sites of wild olives, O. europaea nr. ssp. cuspidata, in the Western Cape Province, South Africa, where a total of 252,603 unripe and 139,872 ripe wild olive fruit were collected (Table 2). Unripe fruit was significantly smaller (pulp thickness = 0.94 ± 0.04, n = 32) (F 1,73 = 169.4, p < 0.001) than ripe fruit (mean ± SE pulp thickness = 1.93 ± 0.06, n = 43). Mean monthly host density (or fruit infestation rate) on unripe and ripe fruit were 4.5 ± 0.7% (range 0.5-11.7%) and 14.8 ± 2.0% (range 4.6-41.2%), respectively (Fig. 5A). Host density increased with fruit maturity and was affected by the interaction between fruit maturity and seasonal tempera-Figure 1. Composition and relative abundance (%) of braconid parasitoid species reared from Bactrocera spp. on wild olives in seven regions of Africa; small red circles show approximate locations of sampling sites and the parasitoid composition size is proportional to the number of fruits (n) collected in each region. The map was created in R (version 3.6.3) using 'rworldmap' package (version 1.3.6, https ://cran.r-proje ct.org/web/packa ges/ rworl dmap/rworl dmap.pdf) 69 (Table 3). Most emerged flies were B. oleae; only 0.87 ± 0.39% (mean ± SE) and 1.90 ± 0.66% (n = 24) of the emerged flies from the unripe and ripe fruit were B. biguttula. Combined parasitism of B. oleae and B. biguttula were 28.6 ± 2.8% (mean ± SE) and 25.4 ± 2.6% on the unripe and ripe fruit, respectively. Parasitism levels were not associated with fruit maturity but rather negatively related to mean temperature (Table 3), decreasing only during mid-summer months (Fig. 5B). All four braconid parasitoids, B. celer, P. humilis, P. lounsburyi and U. africanus, were found in both unripe and ripe fruit. Diversity was generally lower in unripe than ripe fruit (Fig. 6A). Utetes africanus was the predominant parasitoid, followed by P. lounsburyi while both P. humilis (mean 1.8% and 2.5% on the unripe and ripe fruit, respectively) and B. celer (mean 0.1% and 4.9% on unripe and ripe fruit, respectively) were less common in both unripe (Fig. 6B) and ripe (Fig. 6C) fruit. GLM analyses showed that diversity was not affected by mean temperature but was positively related to fruit maturity and host density. The relative abundance of U. africanus was affected negatively by fruit maturity and the presence of other parasitoid species but was positively related    Table 4). The relative abundance of P. lounsburyi was also affected positively by fruit maturity, seasonal temperature, and host density but negatively by the presence of other parasitoids (Table 4).  The biplot shows the relationships between bioclimatic variables and dominance of parasitoid species in each region. Climatic predictors are: Ann tem annual mean temperature, Max tem maximum temperature of the warmest month, Min tem minimum temperature of the coldest month, Warm tem mean temperature of the warmest quarter, Cold tem mean temperature of the coldest quarter, Ann prec annual precipitation, Wet prec precipitation of the wettest quarter, Dry prec precipitation of the driest quarter, and the relative proportion of P. lounsburyi (P.l.%), P. humilis (P.h.%) and U. africanus (U.t.%) to emerge from parasitized fruits. PCA statistics and graphs were performed using JMP Pro ver13 (SAS, Cary, NC).

Discussion
We conducted the largest modern exploration for olive fruit fly parasitoids in Africa. Our surveys reveal remarkable differences in distribution, diversity and dominance of braconid parasitoid guilds from wild olives across the African continent. The sub-Saharan regions of Namibia, South Africa and Kenya maintained the highest diversity of braconid B. oleae parasitoid species, supporting the argument of a Sub-Saharan origin of B. oleae [22][23][24] . We found only one native braconid parasitoid (P. concolor) in northern Africa, despite climates in the sampled regions being similar to that of Namibia. We recovered only D. sp. near fullawayi on Réunion Island, where the native Afrotropical species Diachasmimorpha fullawayi (Silvestri) was reported from other tephritid fruit flies 41 .
No braconid parasitoids are reported to occur naturally in Europe's Mediterranean Basin 25,26 or California 40 . Although P. concolor was found in Corsica, France, where this parasitoid has never been released, it was uncertain if it was accidently introduced from established regions in Italy or North Africa 27 . Although not discussed here, surveys in Asia were also conducted in India, Nepal, Pakistan and China, and from these collections another braconid parasitoid, Psytallia ponerophaga (Silvestri), was reared from B. oleae in Pakistan and D. longicaudata (Ashmead) was recovered in China 21,42 . All five parasitoids reared from B. oleae are larval parasitoids and four of them (D. sp. near fullawayi, P. humilis, P. lounsburyi and U. africanus) are koinobiont endoparasitic opiine wasps; B. celer is an idiobiont ectoparasitic braconine wasp [43][44][45] . Among tephritid fruit fly parasitoids, only a few are braconine parasitoids and nearly all of these are idiobiont ectoparasitoids of the larval flies 17 . No egg parasitoids of B. oleae were found in the current survey reported herein, or in previous surveys 12,30,32,33,46 , although one generalist egg parasitoid, Fopius arisanus (Sonan) (Hymenoptera: Braconidae), was able to attack and develop from B. oleae under quarantine conditions 47 . In our collections, parasitoids were obtained from pupae collected after exiting fruit or by rearing adults from infested fruit. It is likely that parasitoids that locate and attack hosts in the soil after larvae drop from fruit, or following pupation, have been underrepresented 46 . Some pupal parasitoids such as Pachycrepoideus vindemiae Rondani (Hymenoptera: Pteromalidae) were known to attack B. oleae 30 . Other chalcidoid parasitoid species were reported previously from Africa attacking fruit flies in multiple families, and they are considered to be relative generalists and would not be recommended for introduction for biological control e.g., 48,49 . The other two closely related fly species recovered, B. biguttula in South Africa and B. munroi in Kenya, were also collected from wild olives. The collected parasitoids may also attack these two fly hosts, but B. oleae is thought to be their major host species as the number of the other two fly species were extremely low in South Africa and Kenya and not recovered in Namibia during our collections.    11,50 , which may impact their establishment in regions with either hotter summers or colder winters. Although little is known about U. africanus' temperature tolerance, the current surveys showed U. africanus was more abundant in the Mediterranean-like climates. Many other biotic and abiotic factors could also affect the distribution of these parasitoids. Rainfall patterns would strongly influence the seasonal occurrence and abundance of fruit availability, and consequently the abundance of flies and their parasitoids. In drier habitats, the fruit is likely to be small and ripen slowly, offering little food for fly larvae. Annual precipitation was consistently highest in Kenya, as were olive fly populations and their parasitoids (Table 1). Interspecific competition may occur and coexistence between these species is likely facilitated by niche segregation through differentiation in biological or ecological traits. As shown in South Africa, www.nature.com/scientificreports/ U. africanus was more dominant on small and unripe fruit whereas P. lounsburyi was more dominant on ripe fruit. Large ripe fruit may limit the access of U. africanus, which has the shortest ovipositor among all five larval parasitoids 10 , but other parasitoids such as P. lounsburyi fill the host feeding niches. If interspecific competition shapes the parasitoid guilds, it likely would show a similar dominance across different regions. Thus, adaptation to abiotic conditions is likely a major force underpinning diversification and dominance of these species. The four braconid parasitoids recovered in sub-Saharan regions (B. celer, P. humilis, P. lounsburyi and U. africanus) have been imported and evaluated in classical biological control of B. oleae in California 51 , whereas D. sp. near fullawayi was not evaluated. In addition, D. longicaudata was also found to readily attack B. oleae 10,43,52 , but it is a generalist parasitoid of tephritids 41 . Among the well-adapted African braconid parasitoids, the relatively shorter length of U. africanus ovipositors match with lower pulp thickness of wild olives. This parasitoid is ineffective on cultivated olives that has higher pulp thickness through breeding programs. This thicker pulp allows B. oleae fly larvae to move deeper into the olive pulp to escape attack from larval parasitoids that have short ovipositors 10,52 . Bracon celer is able to attack the Cape ivy fly, Parafreutreta regalis Munro (Tephritidae: Tephritinae), which itself was introduced from South Africa into California for the control of the invasive Cape Ivy weeds 43,53 . Psytallia concolor is also a common parasitoid of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in eastern Africa 12,41,54 . Although the current surveys did not find it on B. oleae in Kenya, it has been previously collected from coffee-infesting C. capitata in other parts of Kenya 54 . Genetic analysis showed clear separation of the North African populations from the Sub-Saharan populations and thereafter referred the Sub-Saharan P. concolor populations (often described as P. cf. concolor 54,55 ) as P. humilis 56,57 . Psytallia lounsburyi has been reared only from B. oleae 41,54,58,59 and is the most host-specialized parasitoid among all parasitoid candidates 60 . In the Mediterranean Basin, P. concolor is the only parasitoid that has been extensively studied e.g., 61 and widely released with partial establishment in the southern regions 33,62 .
The current study also showed that fruit maturity, seasonal variations in climates and interspecific competition likely shape seasonal host-parasitoid dynamics in South Africa. Collectively, the co-adaptation of parasitoids and hosts may have contributed to maintaining low host population densities in its native range where fruit infestation rate was generally less than 15%. In contrast, untreated olives can reach 100% infestation in California 63 . Although olive fruit fly larvae are not tolerated in fruit used for canning, 10-30% infested fruit can be tolerated in olives that are pressed for oil in California. In California, two P. humilis populations, originated from B. oleae on wild olives in Namibia and C. capitata on coffee in Kenya, were released without subsequent establishment 40,[64][65][66] . However, two populations of P. lounsburyi, originating from Kenya and South Africa, were released and successfully stablished along the California coastal regions 40 . Low winter survival may contribute to the failure of establishment of P. concolor in northern Mediterranean Basin 62 and P. humilis in California 11,67 . For this reason, P. ponerophaga from Pakistan is being considered for release in California as it was found to have higher rates of low temperature survival than P. humilis 68 . Other factors such as availability of olive fruit for the host-specific B. oleae and alternative hosts could also restrict the establishment of its specialized parasitoids in introduced regions. In South Africa, wild olives are widely available all year, and alternative hosts may also help parasitoid populations to survive periods when local B. oleae populations are sparse 31,54 .
In conclusion, this study reveals the diversity, geographical distributions and dominance of parasitoids closely associated with B. oleae in the fly's native range and provides a better understanding of potential ecological factors that may affect the efficiency and establishment of candidate parasitoids as biological control agents. In particular, identifying the suitable climatic niche of these different parasitoid species and understanding their geographic Table 4. Generalized Linear Model testing the effects of (1) fruit maturity (unripe vs. ripe), mean monthly temperature and host density (= % fruit infested) on diversity of braconid parasitoids, and (2) fruit maturity, mean monthly temperature, host density, and incidence of other braconids on the relative abundance of the dominant braconids U. africanus or P. lounsburyi in Western Cape, South Africa. www.nature.com/scientificreports/ predictability helps to determine the potential establishment in released habitats in the presence of biotic interactions is paramount for successful biological control of B. oleae. Alteration of fruit morphological traits (such as size) through domestication may modify the tri-trophic interactions in agricultural eco-systems, reducing the efficiency of the larval parasitoids with short ovipositors on cultivated olives 10,43,52 . Therefore, understanding both the ecological niche and the co-evolutionary history of the host and parasitoid is fundamentally important for effective classical biological control. The recent successful establishment of P. lounsburyi in California should evoke further investigation into the use of this species for classical biological control of B. oleae in other climatically similar regions 31,46,51 . The advances of modern rearing techniques for these exotic parasitoid species and their tephritid hosts should further facilitate the use of classical and augmentative biological control of B. oleae 37 .

Materials and methods
Regional exploration. African collections for B. oleae and its parasitoids were conducted from 2000-2011 in seven regions: parts of Kenya, Namibia, South Africa, the Canary Islands, Tunisia, Morocco and Réunion Island ( Fig. 1; for detailed locations of collection sites see Fig. S1) Collected fruits were kept at room temperature (20-23 °C) in collaborating laboratories or hotel rooms near collection sites. The fly larvae often pupate inside unripe fruit but will exit and pupate outside of ripe fruit (typically in the soil underneath the tree in situ). When available, the majority of collected fruit were ripe, this allowed an easier collection of the fly puparia emerging from fruit, although both unripe and ripe fruit were collected. Larval ectoparasitoids, such as B. celer, emerge directly from fruit, which were held for up to one month for maximum emergence of flies or parasitoids. When possible, the emerged pupae were returned with the collectors or sent by cooperators to the ARS European Biological Control Laboratory (EBCL), otherwise the material was held at collaborating laboratories for emergence of flies or parasitoids.
All emerged insects were identified to species and gender. All parasitoid species were initially identified and/ or subsequently confirmed by Dr. Robert Wharton (retired, Texas A&M university). Keys of these fruit fly parasitoids are available at http://paroffi t.org 17,38 . These parasitoid species were further confirmed through molecular analyses 21,53 . Most of these parasitoid species can be easily identified based on the morphological characteristics as described by 17 , except for P. humilis and P. concolor; these two species are morphologically indistinguishable [52][53][54] . However, genetic analysis showed a clear separation of the north African and sub-Saharan populations 53 . According to Rugman-Jones et al. 53 , P. humilis is the attributed name for all sub-Saharan populations while P. concolor is the attributed name for all north African populations. The relationship of these two species was also confirmed in other studies 44,45 (Fig. 7). These sites were located within 200 km of each other and ranged in elevation from 77-823 m. Approximately 900 fruits of wild olive were collected at each site once every 2-4 weeks, depending on the availability of fruit. Collected fruit were processed at Stellenbosch University and sorted by size and condition. Fruit size, or pulp thickness, was assumed to affect some parasitoid species ability to find and oviposit into fly larvae feeding deeper inside the fruit because of their short ovipositors 10,11 . Fully ripe (black) fruit is generally larger than unripe (green) fruit and fly larvae will feed deeper inside the softer fruit. Therefore, green and black fruit were sorted and assessed separately. Subsamples of unripe and ripe fruit were measured to estimate the pulp thickness of each fruit by inserting an insect pin trough the pulp to the seed three times at randomly selected points on the fruit. The mean depth (pin length minus the exposed portion of the pin) of the three measurements was used to estimate fruit pulp thickness. Collected fruit and emerging puparia were kept at room temperature (20-23 °C) until the emergence of wasps and flies. Data analysis. The relative abundance of each parasitoid species (i.e., percentage of each parasitoid species emerged), total parasitism by all parasitoids and diversity were estimated for each sample in each site and region. Total parasitism was calculated by dividing the total number of emerged parasitoids by the sum of the number of emerged parasitoids and flies (hereafter referred to as apparent parasitism, as the initial mortality was unknown). The Shannon index (H) was used to estimate the diversity: where pi is the proportion of each parasitoid species. Sex ratio (% females) of each parasitoid species was pooled from different regions because initial analyses for each parasitoid species separately did not detect significant differences for any parasitoid among different regions (Table S1). Mean apparent parasitism and diversity among different regions and sex ratio among different parasitoid species were compared using one-way ANOVA. All data were first inspected for normality and error variance for homoscedasticity and all percentage data were logit transformed as needed before analysis. Principal Component Analysis (PCA) was conducted to compare climate among the sampled regions (Tunisia was excluded due to the small samples and its climatic similarity to Morocco) and to analyze potential relationships among regional dominance of parasitoid species and bioclimatic variables in the three Sub-Saharan countries. A set of eight bioclimatic variables were selected for the analyses: annual mean temperature (Ann tem), maximum temperature of the warmest month (Max tem), minimum temperature of the coldest month (Min tem), mean temperature of the warmest quarter (Warm tem), mean temperature of the coldest quarter (Cold tem), annual precipitation (Ann prec), precipitation of the wettest quarter (Wet prec) and precipitation of the driest quarter (Dry prec). These bioclimatic variables were extracted from the WorldClim Global Climate Database 1.3 (http://www.world clim.org) using the R 3.1.3 release. These variables are considered biologically relevant and used commonly in species distribution studies. A biplot analysis was conducted to characterize the relationships.
For the analyses of seasonal host-parasitoid dynamics in South Africa, host fly density was estimated as the number of fly puparia per fruit. Because wild fruit is smaller than cultivated fruit, each wild fruit supports fewer flies, commonly one fly larvae per fruit 46 ; therefore, the fly density per fruit approximately matches the percentage of infested fruit (i.e., fruit infestation rate). Data were pooled from different sites to estimate monthly mean host density or fruit infestation rate, total apparent parasitism by all braconid parasitoids, parasitoid diversity, and the relative abundance of each braconid parasitoids on unripe and ripe fruit, respectively. Linear mixed models were used to analyze the effects of fruit maturity (unripe vs. ripe) and seasonal climate (both were fixed effects) as well as year (random effect) on monthly host density and total parasitism. Host density data were square transformed while parasitism data were logit transformed as needed to meet normality and error variance for homoscedasticity. Monthly mean temperature was used to represent a seasonal climate variable as precipitation was considered similar within the surveyed areas and other temperature parameters (e.g., maximum or minimum temperature) are highly correlated with the mean temperature. The temperature data were obtained from Weather Information (https ://us.world weath eronl ine.com/) from the closest cities (Stellenbosch, Paarl, Citrusdal, Cape Town, Bonnievale or Wellington) of the sampled sites. Generalized linear models (GLM) were applied to analyze the effects of (1) fruit maturity, mean monthly temperature and host density on diversity, and (2) fruit maturity, mean monthly temperature, host density, and incidence of other parasitoids on the relative abundance of two major parasitoids (P. lounsburyi and U. africanus). For GLM analyses, fruit maturity was coded categorically as 1 and 2 for unripe and ripe fruit, respectively, and parasitoid species incidence was coded as 1 (present) and 0 (absent). Percentage data were modelled with binomial distribution and a logit link function while the diversity H = pi ln pi