Morphological Genitalia Variation in Eudocima Phalonia
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Morphological genitalia variation in Eudocima phalonia Catherine Terrell Department of Entomology - Purdue University - West Lafayette, IN 47907, USA Abstract An evaluation of morphological variation in geo-referenced E. phalonia specimens from the zoogeographical regions of Indomalaya and Australasia. Thirty specimens were dissected and measured, 14 female and 16 male. No overall variance was recorded but there was a significant difference between Indomalayan and Australasian specimens in clasper lengths and a marginally significant difference in antevaginal spine lengths. One specimen was identified post-dissection to be a different species, E. homaena. A non-morphometric difference that was observed was in corpus bursa thickness, potentially a result of copulation. Introduction families (Davis, 2005). Economically Eudocima phalonia, also known as important host plant crops that E. phalonia Eudocima fullonia (Clerck 1764), Noctua feeds on include citrus, apples, pears, dioscoreae (Fabricius 1775), Phalaena melons, tomatoes, and strawberries. The Noctua pomona (Cramer 1776), and larvae tend to feed primarily on the foliage Ophideres obliterans (Walker 1858), is in of wild hosts mainly within the the insect order Lepidoptera and in the Menispermaceae and Fabaceae families, but family Erebidae, previously part of the have also been shown to feed and develop family Noctuidae (Zilli, 2017). on red apple under laboratory conditions What makes this insect important is (Davis, 2005). Eudocima phalonia was also its ability to pierce fruits with a sclerotized shown to have a preference for sweet proboscis, allowing harmful bacteria and aromatic fruits compared to those with low fungi to be introduced. There are two major sugar content (reference). An adult food types of fruit piercing categories: primary preference index was developed for this and secondary. Primary feeders are able to moth, and the highest rated fruits were pierce through the skin of the fruit, while bananas and brinjal (aka, eggplants) in 1998, secondary feeders find puncture wounds followed by guava and bananas in 1999 caused by primary feeders (Banziger, 1982). (Bhumannavar, 2012). These distinct feeders cause different types Eudocima phalonia is known to be of damage: primary and secondary. Primary present in temperate broadleaf and mixed damage is the actual or potential ability to forests, tropical and subtropical grasslands, pierce intact skin, and secondary damage savannas and shrubs, and tropical and affects the pulp of an already punctured fruit subtropical forests (Davis, 2005). With these (Banziger, 1982). It is thought that all known biomes and previously mentioned primary feeders can also act as secondary host plants, there is a chance that E. feeders and cause the spread of pathogens phalonia could become a pest across about by secondary feeding by picking up bacteria 30% of the continental US (Davis, 2005). and fungi from inoculated fruit, leading to However, the host plants for larval stages rot (Banziger, 1982). are not commonly found in the US and entry Eudocima phalonia has a wide range potential is considered low, as only 5.8% of of host plants, as both larvae and adults, that all interceptions of previously identified contains more than 100 plants over 34 Noctuidae moths to the USA originated in a 1 country known to have E. phalonia (Davis, Dissections started by boiling water 2005). E. phalonia has also been identified in a 250 mL beaker, removing it from the as“particularly mobile” and “being a hot plate, and placing vials with the moth powerful flyer known to undergo abdomen covered by 10% KOH. They were migrations” (Fay, 1999; Zilli, 2017). This kept in the hot water for 5 to 10 minutes so behavior could aid in becoming an invasive the fat body would break apart and the pest in the Americas. exoskeleton would become relaxed. Scales Eudocima phalonia is commonly were brushed off the body with a paint brush confused with other moths in the genus and then an incision was made on the right Eudocima, including E. materna (which has side following the spiracles to avoid cutting occurred in Florida and Texas), E. homaena, any reproductive organs. The fat body was and E. jordani (neither of which have brushed away with the paint brushes to give occurred in the US) (Davis, 2005). They a clear view of the genitalia. The genitalia possess similar body color and patterns, and was then carefully removed and more debris the only certain way to identify one of these were removed in a fresh dissecting tray. moths is through genitalic dissection by a Petroleum jelly was placed onto a dry qualified taxonomist (Davis, 2005). dissecting tray, covered in 90% ethanol, and The goal of this study was to the genitalia placed on top. A watch glass examine variation among geo-referenced was placed over the genitalia to flatten it in specimens collected for Vernon A. Brou Jr. an attempt to keep measurements uniform Increased understanding of variation in E. during imaging. phalonia would aid quarantine scientists The reproductive parts measured engaged in the process of identification. were the claspers, juxta, and uncus in the Being able to identify this moth if it reaches male and for the females the antevaginal the US will be crucial to inhibit its spread, spine and corpus bursa were measured and knowing any potential variation will looking at the length and width for all. All only benefit this prevention. images and measurements were taken on a M165M Leica microscope for uniformity. Materials and Methods The specimens used for these Results dissections were collected in the Principal Component Analyses Indomalayan and Australasian (PCA), using JMP SAS ver. 14.2.0, were zoogeographic areas, (see fig. A). They were calculated on the male and female collected from as early as 1988 and by specimens, looking at all genital parts various collectors acting for Vernon measured, for males (M): clasper length, Antoine Brou Jr. They were stored in a clasper width, juxta length, juxta width, and freezer between handling, to avoid mold uncus length; for females (F): antevaginal forming on any of the specimens and spine length, antevaginal spine width, prevent its spread on specimens that had corpus bursa length, and corpus bursa width; been affected by mold previously. and comparing Indomalayan specimens to The materials used for dissections Australasian specimens. The levels of were glass vials, 10% KOH, a 250 mL determining significance were: significant beaker, acetic acid, camel hair paint brushes, p-value = <0.05 and marginal significant iris scissors, petroleum jelly, watch glasses, p-value = <0.10. There were no and a M165C Leica imaging microscope. subgroupings found in either males or 2 females, (see fig. B and C). For the male Discussion PCA results, there was one clear outlier so a There were no major overall test was run to locate any additional outliers variances found in the genitalia of E. (see fig. D). Once those outliers were phalonia, from across the zoogeographic removed (three in total), a second PCA was regions of Indomalaya and Australasia. run on the male moths again and component Previous studies have found several biotypes 2 (composed of 0.736 eigenvector as clasper within E. phalonia that need evolutionary length) gave a marginally significant p-value value assessments, such as between of 0.0896 (see fig. E). This prompted Afrotropic and Indoaustralian specimens further investigation into the data to find if (Zilli, 2017). Our current study would imply any individual parts were significantly that all moths across this area (Indomalaya different between Indomalayan and plus Australasia) have been selected for the Australasian specimens. To accomplish this, same sets of traits across these areas. T-tests using Microsoft Excel 2016 were Further investigation of Afrotropical performed. From these tests there was one specimens is needed for this lack of marginally significant different part: variation across the entire species to be antevaginal spine length (t(5) = -1.681, p = confirmed. 0.07) (see fig. F and fig. G). Additionally, Small variation is visible, however, there were differences in corpus bursa when looking at individual genital parts. shape, but these were not quantified as it is a Looking more beyond the qualitative results complex, non-linear shape (see fig. H and at the corpus bursae shape in female moths, fig. I). Finally, one male moth stood out some had thicker mid sections than others, from the other male moths. Further (see fig. H and I. fig. H). Thicker corpus investigation showed that it was bursae show that there are cornuti present in misidentified as E. phalonia, (see fig. J) but the bursa, which could indicate that there are after dissection this specimen was found to physiological differences between mated be E. homaena (see fig. K). and unmated female moths. Further investigation is also needed for this to be confirmed. 3 fig. A Map of commonly used zoogeographic regions where individuals are more similar to each other within regions rather than outside. fig. B fig. C Principal component analysis of male moth genitalia Principal component analysis of female moth genitalia comparing different reproductive parts for Indomalayan comparing different reproductive parts for Indomalayan and Australasian specimens. The first parameter estimates and Australasian specimens. The first parameter estimates is for component 1 and the second is for component 2. is for component 1 and the second is for component 2. 4 fig. D fig. E Outliers found using JMP SAS. Principal component analysis of male moth genitalia comparing different reproductive parts for Indomalayan and Australasian specimens after outliers were taken out. The first parameter estimates is for component 1 and the second is for component 2 fig. F Bar graph showing standard error means for male reproductive parts for zoogeographical regions. 5 fig. G Bar graph showing standard error means for female reproductive parts for zoogeographical regions. fig. H fig. I Female reproductive organs (corpus bursa and antevaginal spine present) showing qualitative difference.