Research Article |
Corresponding author: Mark K. Schutze ( m.schutze@qut.edu.au ) Academic editor: Marc De Meyer
© 2015 Mark K. Schutze, Thilak Dammalage, Andrew Jessup, Marc J.B. Vreysen, Viwat Wornoayporn, Anthony R. Clarke.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Schutze MK, Dammalage T, Jessup A, Vreysen MJB, Wornoayporn V, Clarke AR (2015) Effects of laboratory colonization on Bactrocera dorsalis (Diptera, Tephritidae) mating behaviour: ‘what a difference a year makes’. In: De Meyer M, Clarke AR, Vera MT, Hendrichs J (Eds) Resolution of Cryptic Species Complexes of Tephritid Pests to Enhance SIT Application and Facilitate International Trade. ZooKeys 540: 369-383. https://doi.org/10.3897/zookeys.540.9770
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Laboratory-reared insects are widely known to have significantly reduced genetic diversity in comparison to wild populations; however, subtle behavioural changes between laboratory-adapted and wild or ‘wildish’ (i.e., within one or very few generations of field collected material) populations are less well understood. Quantifying alterations in behaviour, particularly sexual, in laboratory-adapted insects is important for mass-reared insects for use in pest management strategies, especially those that have a sterile insect technique component. We report subtle changes in sexual behaviour between ‘wildish’ Bactrocera dorsalis flies (F1 and F2) from central and southern Thailand and the same colonies 12 months later when at six generations from wild. Mating compatibility tests were undertaken under standardised semi-natural conditions, with number of homo/heterotypic couples and mating location in field cages analysed via compatibility indices. Central and southern populations of B. dorsalis displayed positive assortative mating in the 2010 trials but mated randomly in the 2011 trials. ‘Wildish’ southern Thailand males mated significantly earlier than central Thailand males in 2010; this difference was considerably reduced in 2011, yet homotypic couples from southern Thailand still formed significantly earlier than all other couple combinations. There was no significant difference in couple location in 2010; however, couple location significantly differed among pair types in 2011 with those involving southern Thailand females occurring significantly more often on the tree relative to those with central Thailand females. Relative participation also changed with time, with more southern Thailand females forming couples relative to central Thailand females in 2010; this difference was considerably decreased by 2011. These results reveal how subtle changes in sexual behaviour, as driven by laboratory rearing conditions, may significantly influence mating behaviour between laboratory-adapted and recently colonised tephritid fruit flies over a relatively short period of time.
Oriental fruit fly, sexual compatibility, isolation indices, laboratory adaptation
While now debated as to whether it is a driver of speciation, or a secondary effect flowing from population divergence (
For tephritid fruit flies, cross-species mating in small cages results in forced matings that produce viable hybrids (
International protocols for tephritid mating trials were initially designed to test competiveness and compatibility among flies mass-reared for the sterile insect technique (SIT) and their wild counter-parts, or to compare the competitiveness and compatibility of populations from different mass-rearing facilities in different geographical areas (
In this paper, we report on two crossing experiments using the same populations of Bactrocera dorsalis (Hendel), conducted under identical experimental conditions exactly 12 months apart. The first cross used nearly wild flies (F1-F2 generation), while the second cross used flies from the same colony when six generations in culture (F6). Mating compatibility between the two populations was assessed between the trials, and this example was used to discuss: i) the importance of collecting secondary behavioural data in mating trials; ii) the importance of understanding subtle differences in courtship behaviour which may occur between wild populations of the same species; and iii) the implications of using ‘wildish’ versus laboratory-adapted populations for integrative taxonomic research.
We evaluated mating behaviour of B. dorsalis from central and southern Thailand. All flies were sourced directly from the wild via host-rearing and sent to the FAO-IAEA Insect Pest Control Laboratory (IPCL), Seibersdorf Austria, in March 2010. Collection locations were not privately owned and no endangered or protected species were involved in the study. No specific permits were required for the described field studies or for the import of live material into the IPCL. The central Thailand population was reared from Mangifera indica L. (Anacardiaceae) in Saraburi and sent as a batch of approximately 500 pupae; the southern population was reared from Carica papaya L. (Caricaceae) in Nakhon Si Thammarat and sent as a batch of approximately 200 pupae.
Flies were morphologically examined for external and internal genitalic characters to confirm their identity in accordance with taxonomic descriptions (
Adult flies were provided a standard diet of enzymatic yeast hydrolysate and sugar (1:3) together with water ad libitum. Sexually mature flies were exposed to egg-cups dosed with commercial guava juice (Rubricon, Rubricon Products, Middlesex, U.K.) as an oviposition stimulant. Eggs were incubated overnight (25 ± 2 °C, 65% R.H.) on moist filter paper placed on wet sponges in Petri dishes and then transferred to carrot diet (
The first mating compatibility tests were conducted in June and July of 2010 when Saraburi and Nakhon Si Thammarat colonies were at the F1/F2 laboratory colony generation. Eight replicates were completed, consisting of five using F1 generation flies and three using F2 generation flies. The second series of mating compatibility tests were undertaken one-year later in July 2011 when both cultures had reached F6 (eight replicates completed). Experimental protocols were identical for 2010 and 2011 trials, as outlined below.
Flies were sexed within four days of emergence; this is well before male and female sexual maturation which occurs 15-20 days post emergence based on personal observation (MKS; data not shown) and previous studies (
Field cage tests were conducted inside a glasshouse exposed to natural light and maintained at ~25 °C and ~50% R.H. Replicates were undertaken inside one of four partitioned flight cages (2.0 m × 1.6 m × 1.9 m) within the glasshouse, with each cage containing a single, non-fruiting potted Citrus sinensis Osbeck (Rutaceae) tree of 2 m in height with a canopy of ~ 1.1 m in diameter.
Flies were released into the experimental field cage at a 1:2 male:female ratio. As this study was focussed on mating compatibility and not strictly competition, this ratio of males to females (as opposed to 1:1) was used to ameliorate the effect of potentially early-mating males from monopolising all females from one population and thus inflating isolation indices, as per
Relative percentages of each of the four possible couples (i.e., Saraburi ♂ × Saraburi ♀ [SS], Saraburi ♂ × Nakhon Si Thammarat ♀ [SN], Nakhon Si Thammarat ♂ × Saraburi ♀ [NS], and Nakhon Si Thammarat ♂ × Nakhon Si Thammarat ♀ [NN]) were calculated for each replicate. Proportion data were arcsine transformed prior to subsequent analysis; one-way anova (with Tukey post hoc test where appropriate) was conducted to assess for significant differences among mating combinations within each year; paired t-tests were conducted to assess for significant differences in relative proportions of respective couple combinations across years.
Compatibility was determined using the Index of Sexual Isolation in conjunction with the Male Relative Performance Index and the Female Relative Performance Index (
Ninety-five percent confidence intervals of the used indices (ISI, MRPI, and FRPI) for each of 2010 (F1/F2 flies) and 2011 (F6 flies) were calculated to determine deviations from random mating (ISI = 0) or equal participation by the respective sexes (MRPI & FRPI = 0). Confidence intervals that included zero represent cases of random mating and equal participation between the populations. Heterogeneity chi-square analyses across replicates for each treatment were undertaken to determine if data could be combined prior to further analysis. Following heterogeneity tests, chi-squared tests of independence were applied to determine if males mated predominantly with females of one population over the other.
The mean time to begin mating (mating latency) was estimated by calculating how many minutes had elapsed between the time each couple initiated mating and the time of the first observed mating couple (= time zero) within each particular cage replicate (as per
All values reported represent mean ± 1 s.e. unless otherwise stated.
Eight replicates of mating compatibility tests were completed for each of the 2010 and 2011 trials; 84.7 ± 2.1% and 78.1 ± 4.3% flies mated in 2010 and 2011, respectively.
Total numbers and mean percentages of each of the four possible mating-pair combinations (i.e., SS, SN, NS, and NN) varied considerably between years (Figure
Behavioural parameters of Bactrocera dorsalis flies from Saraburi (S) and Nakhon Si Thammarat (N) (Thailand) during mating compatibility trials in 2010 and 2011. A Relative percentages and total numbers of each possible couple formed. Numbers in bars are total numbers of each couple formed summed across replicates B Mating latency as average time since first couple observed for couples formed C Average percentage of respective couples collected from the tree for each of the six mating compatibility comparisons. For all graphs, columns surmounted by the same letter within a year are not significantly different at α = 0.05.
Analysis of latency (time to mate since first couple formed) revealed further differences between populations of B. dorsalis from Nakhon Si Thammarat and Saraburi (Figure
There were no significant differences among mating combinations with respect to position on the tree or cage wall for the 2010 trial (F(3,28) = 0.134, p = 0.939); however, there was a significant difference among couples in the 2011 trial (F(3,28) = 3.902, p < 0.05) (Figure
As chi-squared tests of independence were homogeneous across replicates for both years (2010 χ2 = 3.49, df = 7, p = 0.836; 2011 χ2 = 11.18, df = 7, p = 0.131), data were summed prior to analysis of mating indices ISI, MRPI and FRPI. While there was a significant bias towards assortative mating in 2010 (χ2 = 13.64, df = 1, p < 0.0001; ISI = 0.26 ± 0.19 [95% C.I.]), this effect was largely lost by the time F6 flies were crossed in 2011, despite the consistent and significant increase in number of NN couples (χ2 = 2.32, df = 1, p = 0.128; ISI = 0.11 ± 0.10 [95% C.I.]) (Figure
Index of Sexual Isolation (ISI) and relative performance indices for males (MRPI) and females (FRPI) with associated 95% confidence intervals calculated for 2010 and 2011 mating compatibility comparisons between Bactrocera dorsalis from Saraburi and Nakhon Si Thammarat, Thailand. Dotted line (0.00) represents random mating (ISI) or equal participation by the sexes (MRPI and FRPI).
The FRPI significantly deviated from random (FRPI = -0.37 ± 0.12 [95% C.I.] in 2010, reinforcing that considerably more Nakhon Si Thammarat females mated (n = 186 summed across reps for SN and NN) relative to those from Saraburi (n = 85 summed across reps for SS and NS). While this trend continued in 2011, there was a considerably reduced difference in female participation (n = 140 versus n = 110, resp.) as reflected in the FRPI measure approaching zero (FRPI = -0.13 ± 0.12 [95% C.I.]). While less dramatic, there were also significantly more males from Nakhon Si Thammarat mating (n = 153 summed across reps for NS and NN) relative to those from Saraburi (n = 118 summed across reps for SS and SN) in 2010 with a mean MRPI (± 95% C.I.) of -0.13 ± 0.05 (Figure
Our results show that F1/F2 (= ‘wildish’) B. dorsalis from Saraburi and Nakhon Si Thammarat demonstrated significant positive assortative mating: i.e., like was more likely to mate with like than expected by chance. This assortative mating was lost by the 6th generation, when random mating occurred between the two populations. The change from positive assortative to random mating was most likely due to two factors: latency and relative participation of the sexes. In ‘wildish’ populations Nakhon Si Thammarat males mated sooner than Saraburi males (i.e., their mating latency time was shorter) and Nakhon Si Thammarat females mated more than Saraburi females. The combination of the two attributes led to more Nakhon Si Thammarat × Nakhon Si Thammarat matings. By the 6th generation, the temporal difference in male latency was lost, as was the increased ‘precociousness’ of the Nakhon Si Thammarat females, leading to random mating between the populations.
Differences in latency in male mating behaviour may be the results of local environmental conditions from where respective populations of B. dorsalis originated. Time of sunset, for example, may be a potential causal factor, considering Nakhon Si Thammarat is located approximately 600 km south of Bangkok and time of sunset (and time of mating) would correspondingly vary. However, despite their geographic distance, time of sunset differs little between these locations across the year: the sun sets approximately 10-12 minutes later in Nakhon Si Thammarat relative to Bangkok in January, yet in July it sets approximately 6 minutes earlier (based on 2014 sunset data; www.sunrise-and-sunset.com). Nevertheless, this slight difference may be sufficient to influence mating latency in early-generation laboratory colony flies. Complexity in circadian rhythm patterns and differences in mating latency between wild and mass-reared colonies have been investigated in other tephritid species, such as the melon fly Bactrocera cucurbitae (Coquillett) (
What influenced variation in female mating in our trials remains open to speculation. Drivers of sexual propensity are varied, and may include both intrinsic and extrinsic (e.g., temperature, food, density, and sex ratios) factors (
These results pose a conundrum for mating trials. It is generally considered that the use of ‘wildish’ populations (i.e., within one or very few generations of field collected material) is more desirable than using flies that already have been cultured for a long time because laboratory selection may alter key behavioural and physiological traits (
Based on this experiment alone we are not in a position to make strong statements about using ‘wildish’ versus older cultures for mating tests in B. dorsalis, but we do highlight that even within a single biological species, local adaptation and drift may lead to subtle but potentially important differences in some aspects of the mating system, as documented in other organisms (
As a factory quality assurance measure, mating indices serve a valuable function by allowing repeatable and quantifiable measures of the quality of factory flies, thereby forming an effective tool allowing SIT action programme managers to determine if sterile males are fit for purpose to compete with wild males and are compatible with wild females. But there is no doubt that these indices, by focusing exclusively on the ‘end product’ of copulation, may lead some researchers to potentially under appreciate biologically important steps in the courtship process. Where mating is 100% random, or 100% positive-assortative, these prior steps may be less critical for interpreting meaning from the trials. But where the results fall in between these extremes, as has been found in several Bactrocera and Anastrepha studies (
The use of very recently established colony material is widely considered ideal for determining mating compatibility among strains, populations, or putative species. Our results clearly demonstrate that subtle behavioural characteristics may ‘carry-over’ from the wild and may result in inflated measures of incompatibility that are soon lost following colony establishment. For future tephritid research where mating is used to help delimit cryptic species, we therefore encourage the use of detailed courtship behaviour in field cage mating studies that is quantified by isolation and additional indices that dissect specific behavioural attributes among populations or putative species.
We acknowledge the support provided for this research via the FAO-IAEA Coordinated Research Project “Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Applications and International Trade” and the Australian Government Cooperative Research Centre (CRC) Scheme (Project #20115). We also sincerely thank colleagues that assisted in the collection of material for this project.