Identification and Estimation of Instar Number of The Twig Borer Weevil (Coleoptera: Curculionidae) on Albizia, Falcataria moluccana (Miquel) Barneby & J.W. Grimes in Maluku

Abstract

Albizia, or Falcataria moluccana (Miq.) Barneby & J.W. Grimes is known as “sengon” in Indonesian, “jeungjing” in Java, and “salawaku” in Maluku. This tree is native to Indonesia, originating from Maluku and Papua. As a fast-growing tree, F. moluccana is reported to be an invasive species in the lowland humid forests on the Pacific and Indian Ocean islands. In the natural habitat of F. moluccana, the twig borer weevil (Coleoptera: Curculionidae) is found. The weevil is an important pest of F. moluccana and can be used as a candidate for its natural enemies. Information regarding the weevil was not available before. The aims of this study are (1) to study the taxonomy of the Albizia twig borer weevil through morphological and molecular characters in Maluku Province, (2) to descibe sexual dimorphism based on morphological characteristics and measuring the morphometrics of the Albizia twig borer weevil in Maluku Province, (3) to estimate the number of larval instars of the Albizia twig borer weevil in Maluku Province. The study provides information of taxonomy, morphological, morphometric, and molecular characters, as well as estimation of the larval instar numbers of the Albizia twig borer weevil in Maluku Province, Indonesia. This research was conducted from February to December 2022. Samples of weevils and larvae were collected at Albizia plantations which grow naturally around the roadside in Maluku Province, which are spread over three islands: Ambon, Seram, and Haruku Islands. The research was conducted with three main topics: morphological and molecular identification; sexual dimorphism of adults through external body morphology and morphometric measurements, and estimation of larval instar numbers. Morphological identification was conducted using a dichotomous key at the subfamily and tribe levels, and it was further compared and matched with type specimens in the museum. Molecular identification utilized the mtCOI gene, employing the forward primer (3'-GGTCAACAAATCATAAAAGATATTGG-5') and the reverse primer (5'-TAAACTTCAGGGTGACCAAAAAATCA-3'). Morphometry involved measuring 25 females and 21 males on 18 body parts, including BL (total body length), RL (rostrum length), MRL (metarostrum length), RAW (rostrum apex width), RBW (rostrum base width), SL (scape length), FL (funicle length), ProL (pronotum length), ProW (pronotum width), ProBW (pronotum base width), EL (elytra length), EBW (elytra base width), EWMax (elytra maximum width), EWMin (elytra minimum width), EAW (elytra apex width), Fem1L (first femur length), Tib1L (first tibia length), and Tar1L (first tarsus length). Differences in morphometric measurements between males and females were analyzed using a t-test (P < 0.05) on Minitab ver. 21. Principal component analysis (PCA) was used to assess variation in morphometric characters and determine which characters were the most representative in size between males and females on PAST ver. 4.11. Larval instar numbers were estimated by measuring head capsule width at the widest point of the head capsule. Kernel density estimation (KDE) was used to estimate the number and limits of instars. Gaussian mixture model (GMM), a clustering model, was applied to ensure the number of instars derived from KDE. The results of this study were expected to provide important information regarding the Albizia twig borer weevil in Maluku Province, Indonesia and offer basic insights into natural enemies that could be utilized in developing a biological control program for F. moluccana. The weevil was identified as belonging to the tribe Ithyporini and confirmed as a twig borer weevil of F. moluccana. Taxonomically, this weevil belonged to the subfamily Cryptorhynchinae and the tribe Ithyporini. However, it had not been identified to the genus and species level. Scientifically, it could be referred to as the weevil of the tribe Ithyporini, or as Ithyporini gen. sp., or simply as the Albizia twig borer weevil. Morphologically, the Albizia twig borer weevil was almost identical to the genus Perrhaebius F. P. Pascoe 1874 from Java and Sumatra, but there were differences in scutellum, sternulum, and basisternum. Scales were present in the scutellum, sternulum, and basisternum of the Albizia twig borer weevil, but not in Perrhaebius sp. Molecular identification showed that the Albizia twig borer weevil had the highest homology value at 86.41% with Deretiosus aridus from Indonesia. The most pronounced sex difference that could be used to discern sexual dimorphism in external morphology was the rostrum, an elongated cuticular head extension. The surface of the Albizia twig borer weevil’s rostrum, which was shinier and slightly scaled on the anterior side of females, provided a main character that could be reliably used to distinguish consistently and accurately from males. This method was helpful for sex identification in this species without dissection and without destroying the living insects. Based on 18 body character measurements, the morphometrics of the Albizia twig borer weevil could differentiate males and females. Body length, rostrum length, metarostrum length, funicle length, pronotum length, pronotum width, and first tibia length were significantly larger in females than in males. Only the rostrum apex width was significantly larger in males than in females. According to PCA, body length and elytra length were the most representative characteristics in the body size of female and male weevils. Even though the characters overlapped between sexes, rostrum morphology was the most consistent and accurate body structure to distinguish females from males of the Albizia twig borer weevil. This study estimated that the larvae of the Albizia twig borer weevil had four instars during their growth, as indicated by four peaks in the frequency distribution from KDE. Bayesian information criterion (BIC) and Integrated complete-data likelihood (ICL) value in cluster four represented the best-fit model cluster from GMM. This result also agreed with Brooks-Dyar's rule, which stated that the head capsule width grew with a constant geometric of 1.47. In insects, heavily sclerotized structures, such as the head capsule, generally remained approximately the same size within a stadium. Measurable changes in size occurred after moults.

Albizia, atau Falcataria moluccana (Miq.) Barneby & J.W. Grimes dikenal sebagai “sengon” dalam bahasa Indonesia, “jeungjing” di Jawa, dan “salawaku” di Maluku. Pohon ini merupakan pohon asli Indonesia, berasal dari Maluku dan Papua. Sebagai pohon yang tumbuh dengan cepat, F. moluccana dilaporkan sebagai spesies invasif di hutan lembab dataran rendah di pulau-pulau Samudra Pasifik dan Hindia. Di habitat alaminya, kumbang penggerek ranting (Coleoptera: Curculionidae) ditemukan pada F. moluccana. Kumbang ini merupakan hama penting pada F. moluccana dan dapat dijadikan kandidat untuk musuh alaminya. Informasi tentang kumbang ini sebelumnya tidak tersedia. Tujuan penelitian ini adalah (1) untuk mempelajari taksonomi kumbang penggerek ranting Albizia di Provinsi Maluku melalui karakter morfologis dan molekuler, (2) untuk menjelaskan dimorfisme seksual berdasarkan karakter morfologis dan pengukuran morfometrik kumbang penggerek ranting di Provinsi Maluku, (3) untuk memperkirakan jumlah instar kumbang penggerek ranting di Provinsi Maluku. Penelitian ini memberikan informasi terkait taksonomi, karakter morfologis, morfometrik, dan molekuler, serta estimasi jumlah instar larva kumbang penggerek ranting Albizia di Provinsi Maluku, Indonesia. Penelitian ini dilaksanakan dari bulan Februari hingga Desember 2022. Sampel kumbang dan larva dikumpulkan di pertanaman Albizia yang berada disekitar jalan di Provinsi Maluku yang tersebar di tiga pulau: Ambon, Seram, dan Haruku. Penelitian dilakukan dengan tiga topik utama: identifikasi morfologi dan molekuler; dimorfisme seksual kumbang melalui morfologi eksternal tubuh dan pengukuran morfometrik; serta estimasi jumlah instar larva. Identifikasi morfologi dilakukan menggunakan kunci dikotomis di tingkat subfamili dan tribe, serta dibandingkan dengan spesimen tipe di museum. Identifikasi molekuler menggunakan gen mtCOI, dengan primer forward (3'-GGTCAACAAATCATAAAAGATATTGG-5') dan primer reverse (5'-TAAACTTCAGGGTGACCAAAAAATCA-3'). Morfometri melibatkan pengukuran 25 betina dan 21 jantan pada 18 bagian tubuh, yaitu BL (panjang tubuh total), RL (panjang rostrum), MRL (panjang metarostrum), RAW (lebar ujung rostrum), RBW (lebar dasar rostrum), SL (panjang skapus), FL (panjang funikel), ProL (panjang pronotum), ProW (lebar pronotum), ProBW (lebar dasar pronotum), EL (panjang elytra), EBW (lebar dasar elitra), EWMax (lebar maksimum elitra), EWMin (lebar minimum elytra), EAW (lebar ujung elitra), Fem1L (panjang femur pertama), Tib1L (panjang tibia pertama), dan Tar1L (panjang tarsus pertama). Perbedaan pengukuran morfometrik jantan dan betina dianalisis menggunakan uji t (P < 0,05) pada Minitab ver. 21. Analisis komponen utama (PCA) digunakan untuk menilai variasi karakter morfometrik dan menentukan karakteristik yang paling representatif dalam ukuran tubuh jantan dan betina pada PAST ver. 4.11. Jumlah instar larva diestimasi dengan mengukur lebar kapsul kepala di titik terlebar kapsul kepala. Kernel density estimation (KDE) digunakan untuk menentukan jumlah dan batas instar. Gaussian mixture model (GMM), sebuah model pengelompokan, digunakan untuk memastikan jumlah instar yang diperoleh dari analisis KDE. Hasil penelitian ini diharapkan dapat memberikan informasi penting mengenai hama kumbang penggerek ranting di Provinsi Maluku, Indonesia dan memberikan informasi dasar mengenai musuh alami yang dapat digunakan dalam pengembangan program pengendalian hayati untuk F. moluccana. Kumbang ini diidentifikasi sebagai tribe Ithyporini dan dikonfirmasi sebagai kumbang penggerek ranting F. moluccana. Secara taksonomi, kumbang ini termasuk dalam subfamili Cryptorhynchinae dan tribe Ithyporini. Namun, belum diidentifikasi hingga tingkat genus dan spesies. Secara ilmiah, dapat disebut sebagai kumbang dari tribe Ithyporini, atau sebagai Ithyporini gen. sp., atau kumbang penggerek ranting Albizia. Secara morfologi, kumbang penggerek ranting hampir identik dengan genus Perrhaebius F. P. Pascoe 1874 asal Jawa dan Sumatera, namun terdapat perbedaan pada skutelum, sternulum, dan basisternum. Terdapat sisik pada skutelum, sternulum, dan basisternum pada kumbang penggerek ranting Albizia, namun tidak terdapat sisik pada genus Perrhaebius sp. Identifikasi molekuler menunjukkan bahwa kumbang penggerek ranting Albizia memiliki nilai homologi tertinggi sebesar 86,41% dengan Deretiosus aridus asal Indonesia. Perbedaan seks yang paling mencolok yang dapat digunakan untuk membedakan dimorfisme seksual dalam morfologi eksternal adalah rostrum. Permukaan rostrum kumbang penggerek ranting, yang lebih berkilau dan sedikit bersisik di sisi anterior betina, memberikan karakter utama yang dapat digunakan untuk membedakan secara konsisten dan akurat dari jantan. Metode ini berguna untuk identifikasi jenis kelamin pada spesies ini tanpa pembedahan dan tanpa merusak serangga hidup. Berdasarkan 18 pengukuran karakter tubuh, morfometri kumbang penggerek ranting Albizia dapat membedakan antara jantan dan betina. Panjang tubuh, panjang rostrum, panjang metarostrum, panjang funikel, panjang pronotum, lebar pronotum, dan panjang tibia pertama secara signifikan lebih panjang pada betina daripada jantan. Hanya lebar ujung rostrum secara signifikan lebih lebar pada jantan daripada betina. Berdasarkan analisis PCA, panjang tubuh dan panjang elitra merupakan karakteristik paling representatif dalam membedakan ukuran tubuh betina dan jantan. Meskipun karakter-karakter tersebut tumpang tindih antar jenis kelamin, namun morfologi rostrum merupakan struktur tubuh yang paling konsisten dan akurat untuk membedakan betina dan jantan. Penelitian ini menentukan bahwa larva penggerek ranting Albizia memiliki empat instar selama pertumbuhannya, ditunjukkan oleh empat puncak dalam distribusi frekuensi dari KDE. Nilai Bayesian information criterion (BIC) dan Integrated complete-data likelihood (ICL) pada klaster empat paling tinggi, mewakili model kluster terbaik dari GMM. Hasil ini juga sejalan dengan aturan Brooks-Dyar, yang menyatakan bahwa lebar kapsul kepala tumbuh dengan rasio geometris konstan sebesar 1,47. Pada serangga, struktur yang tersklerotisasi, seperti kapsul kepala, umumnya tetap dan berukuran sama dalam satu instar. Perubahan ukuran dapat diukur setelah pergantian kulit.